Physical barriers like head works, dams barrages are main cause of fragmentation and declining of freshwater fish population in natural habitat. Present study focused on RAPD marker technique to assess the genetic variability among and between the populations of endangered sperata sarwari (Singharee) inhabited in the Indus drainage system of Punjab. Total eight populations (80 speciemen) of S. sarwari were collected from the up and downstream of four Rivers (Chenab, Jhelum, Ravi and Indus) of Punjab. Genomic DNA isolated from muscle tissue of adipose side and ten RAPD marker were used, which produced 50 scorable bands with average band ranging from 250–1050 bp which used for further genetic analysis of S. sarwari. Downstream Indus population of S. sarwari showed highest values of observed alleles (na), effected alleles (ne), Nei’s diversity (h), Shannon index (I) and polymorphism which indicated that downstream Indus population was more genetically variant. The genetic variability (0.5124) and genetic flow (0.4758) among the eight population S. sawari was observed. The up and downstream population showed the highest genetic distance (0.5738) and lowest genetic similarities (0.5634) which indicated complete isolation of Ravi population from other six population. Dendrogram showed that up and downstream Ravi population was completely isolated from the other six up and down stream population of S. sarwai. Overall results indicated that the presence of high fragmentation in River Ravi caused the destruction of habitat and decline in population of S. sarwai in the River Ravi.