In this study, we investigated samples from 93 patients with galactosemia in Iran. We identified 20 different mutations in the GALT gene, 8 and 3 mutations in the GALK1 and GALE gene, respectively. Galactosemia is metabolic disorder that can damage the health of a newborn. Screening is an important step to prevent this condition (12). GALT gene located on chromosome 9p13 with approximately 4 kb of DNA sequence arranged into 11 exons with more than 250 pathogenic variants (27). Such pathogenic variants in GALT gene causing loss or severe reduction of GALT enzyme activity can lead to classic galactosemia (28). In this study, patients who carried mutations in GALT gene, showed reduced GALT enzyme activity compared to the normal range so that, it is consistent with study conducted by Garcia et al. (1). In this study we identified 9 new mutations of GALT gene in Iranian population: c.498T > A (P166P), c.529A > G (M177V), c.611G > A (R204Q), c.652C > G (L218V), c.803C > A (T268N), c.876G > A (T292T), c.945T > C (H315H), c.1135G > A (A397T) and c.1128A > T (A376T). Other mutations were previously reported in the studies conducted by Mirzajani et al. (26) and our previous study (13) in Iranian population. In our study, Q188R mutation was found in a compound heterozygous and homozygous state. The p.Q188R mutation was the most frequent mutation in our study (37.7%) so that, this mutation also is the most common mutation in Caucasian (26), Turkish (16), Polish (29), Brazilian (1) and other population (30).The frequency of p.Q188R mutation decreases from west to east across Europe which is in agreement with our study (13). Other most common mutations of GALT gene in our study, was p.Q188R (37/7%), K285N (4.9%), R148W (4.3%) and N314D (3/83%) in return, the R148W, F294Y, R333W, N314D, Q344K and E340K mutations is very common in European and South American population (1, 31). The p.Q188R and p.K285N mutation are common in Caucasian (15) and whites (32). The Q188R, which determines the lack of enzymatic activity and implicitly the most severe form of the disease. Q188R and K285N mutations are common in Eastern European populations, representing 54–70% of classic galactosemia mutations (4, 33). In this study, S135L mutation was found in a compound heterozygous. This mutation is found almost entirely in individuals of African origin, accounting for approximately 48% of African American and 91% of South African GALT mutant alleles (4). In this study, N314D mutation was found as homozygous and compound heterozygous. Heterozygotes for the Duarte allele present 75% of GALT activity, homozygotes present 50% activity, and compound heterozygotes for the Duarte allele and a classic galactosemia allele present 25% activity in RBC. The N314D (the D2 Duarte variant), presented in 5% of the United States population and is associated with the reduction in enzyme activity, causing a mild form of the disease (4, 34). In our study, the K285N mutation, was the second most common mutation so that, this mutation is particularly frequent in countries of Central and Eastern Europe, accounting for 26–34% of galactosemic alleles (3). Heterologous expression of this variant show essentially null activity and homozygosity in patients is associated with essentially null RBC GALT activity and with a severe clinical phenotype (35, 36). Among the new mutations identified in this study, the M177V, R204Q, L218V, T268N and A379T as missense mutations has a higher frequency than synonymous mutations (c.1128A > T, c.945T > C, c.876G > A and c.498T > A). In silico analysis results of the GALT gene show that the most of missense mutations of this gene can cause galactosemia by reducing the stability of the produced protein, changing physicochemical properties and gene interaction. In silico analysis also demonstrate the exist c.945T > C variant as synonymous mutations in GALT gene can change transcriptional regulation by affecting transcription factor binding sites activity or exonic splicing enhancers or silencers, in contrast, the c.1128A > T, c.876G > A and c.498T > A mutation can change exonic splicing enhancers or silencers of GALT gene. GALK deficiency is due to mutations in GALK1 gene (chromosomal location 17q25.1). GALK1 gene is responsible for the catalysis of the first step in glucose metabolism process. The missense mutations in GALK1 protein that lead to galactosemia type 2 (5). In this study we identified 8 new mutations of GALK1 gene in Iranian population: c.593C > T (A198V), c.840 C > T (H280H), c.859C > T (R287C), c.650A > G (K217R), c.852G > T (E284D), c.683G > A (R228H), c.1159G > A (A387T) and c.1152C > T (A384A). In this study, p.A198V mutation was found in a compound heterozygous and homozygous state. The p.A198V (6.44%) mutation was the most frequent mutation in our study. The E284D mutation of GALK1 gene, was the second most common mutation (2/69%) and other missense mutation A387T, R228H, K217R and R287C had a frequency of 2/15% in our study. In the study conducted by Kleopatra et al. (6) the mutation p.Pro28Thr was identified in both alleles in GALK-deficient patients of Roma. In other study performed by Park et al. (37) in Korean population, the three mutation c.-179A > G, c.-27A > C, and c.-22T > C was reported. The important mutations of GALK1 gene which have pathogenic effect including: P28T, A198V, V32M, G36R, R68C, R239Q, T288M, G346S, G349S, and A384P (18), that none of these mutations was found in our study. The A198V mutation of GALK1 gene is designated to be the Osaka variant and was found to be in a significant frequency among the Japanese and Korean populations (19). Our In silico analysis about missense mutations of GALK1 gene showed that exist the A198V, A387T, R228H, K217R, E284D and R287C mutation on this gene, by reducing protein stability, changing physicochemical properties and also changing gene interaction can cause disease. Sneha et al. (5) demonstrated that the A198V alter the structure and function of GALK protein. In silico analysis about two synonymous mutations in GALT gene demonstrate, the c.840 C > T and c.1152C > T mutation of this gene can change exonic splicing enhancers or silencers therefore it can change the function of this gene. Sequencing analysis of the coding region of GALE gene in 93 Iranian patients with identified 3 novel synonyms mutations: c.957G›A, c.879G›C and c.285C > T. The various genetic mutation have been identified in the GALE gene which leads to the occurrence of galactosemia type III. In other study performed by Park et al. (38) in Korean population, the nine mutation p.A25V, p.R40C, p.D69E, p.E165K, p.R169W, p.R239W, p.G302D, p.R335H and p.W336X was reported, that none of these mutations was found in our study. Currently, 22 mutations which result in amino acid changes in the protein have been detected in GALE gene. Of these mutations, only one V94M is associated with the more severe, generalized form of the disease (7). The mutation V180A, is commonly identified but does not appear to be associated with disease (7, 39). The A25V, R40C, D69E, E165K, R169W, R239W and G302D mutation of GALE gene was reported in different studies so that, all these mutations reduces the enzyme activity in the blood and associated with galactosemia type III (7). In silico analysis about three synonymous mutations in GALE gene demonstrate, the c.957G > A, c.879G > C and c.285C > T mutation of this gene can change exonic splicing enhancers or silencers, therefore it can change the function of this gene. The availability of neonatal screening test for galactosemia cause improve clinical outcome. The greatest benefit derived from this approach is that it is possible to introduce therapy before the onset of acute severe complications. Identification of mutations involved in the development of galactosemia in the Iranian population can play an important role in early diagnosis and intervention early in life.