The Sperata (S.) sarwari is one of the commercially important carnivore freshwater fish and is now among the most endangered species in Pakistan. The study was designed to hypothesize that the natural population of S. sarwari has become fragmented due to the construction of barrages and the degradation of natural spawning grounds and also the decrease of genetic diversity is the main cause of decline in population. Molecular markers were applied to assess genetic variations between fish (S. sarwari) populations in four different rivers. Total eight populations were collected from the upstream and the downstream of the River Chenab, Jhelum, Ravi and Indus of the Punjab, Pakistan. The selected morphological and meristic characteristics showed significant (P< 0.05) variations between the upstream and the downstream population and also among the four riverine populations except weight, which was kept constant. For genetic analysis, fifteen RAPD markers were selected from the genomic Library on the basis of GC contents (%) and band reproducibility for the amplification of genomic DNA in S. sarwari. Out of 15 primers, ten RAPD primers produced 50 scorable bands with sizerange of 250-1050 bps. Molecular analysis showed significant polymorphism in the upstream and the downstream of the four riverne populations of S. sarwari. The highest polymorphism (62%) was observed in the River Indus population and lowest level (2%) was observed in the Ravi population. The genetic analysis revealed that the River Indus population showed the high gene diversity (h, 0.253±0.204) and the River Ravishowed the lowest gene diversity (h, 0.0096±0.068). The genetic variability with total heterozygosity (Ht, 0.3574±0.0350) revealed the significant higher variation (Gst, 0.5124) with very low level of migration flow (Nm, 0.4758) that showed no movement of a single individual between the upstream and the downstream of all river populations. Cluster analysis showed significant variations within and among rivers. Furthermore, the dendrogram showed that the S. sarwari population of the River Jhelum and RiverIndus were closely related, while the River Ravi population was completely distinct from other riverine populations. Hence, these genetic analysis described the higher level of genetic variability and lowest genetic flow that increased the inbreeding coefficient in S. sarwari population. Furthermore, this study revealed that the variations in the morphological characteristics are concordance with the genetic analysis. In the present study, it was also concluded that the lowest gene flow is the indication of isolation due to physical barriers i.e. dams and barrage. Hence, this information can be used by Ichthyologist managers and natural conservationist, who are interested in the future to save it from declining of S. sarwari.