Sorghum (Sorghum bicolor) has long posed challenges in In vitro culture due to its recalcitrant nature, leading to limited success with regeneration techniques across various genotypes. This study aimed to assess the influence of hormonal treatments on sorghum callus induction and subsequent regeneration using tissue culture methods. Immature embryos and mature seeds were utilized as explant sources, and varying concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) of the hormones 2,4-D, IAA, BAP, KN, and ZN were employed to induce callus and regenerate six distinct sorghum varieties, namely IS 3477, IS 33095, IS 7005 (non-pigmented), IS 2898, IS 7115, and IS 1202 (pigmented). Callus induction frequencies were markedly distinct among genotypes, with IS 3477 and IS 33095 exhibiting the highest rates, while IS 7115 and IS 1202 showed the lowest. The most significant embryogenic callus induction was observed in IS 3477 (92%) when treated with 2,4-D at 2.5 mg/L and KN at 0.5 mg/L, whereas IS 1202 displayed the lowest induction (48%). Investigation into various cytokinin concentrations revealed that a regeneration medium containing 2.0 mg/L BAP resulted in high regeneration frequencies, particularly in the non-pigmented IS 3477 genotypes, producing the highest number of shoots (20). Furthermore, mature seed explants displayed varying embryogenic callus induction frequencies, with IS 3477 exhibiting the highest (76%) when treated with 2,4-D at 2.5 mg/L and BAP at 0.5 mg/L, while IS 1202 displayed the lowest induction (36%). Optimal regeneration responses were observed with ZN at 1.5 mg/L, with IS 3477 producing the highest shoot count (11). This study elucidated the genotype-specific responses to In vitro protocols across diverse sorghum pigmented and non-pigmented lines. The findings offer valuable insights into the development of high-frequency response profiles, crucial for advancing impending genetic transformation efforts.