Genetic integrity of commercially important fish species is being compromised due to ongoing anthropogenic interventions. Hitherto, there are limited number of studies addressing the questions related to population structure of dwindling fish populations. Studies were undertaken to assess the genetic status of Bagarius bagarius populations from Indus River System by using five microsatellite DNA markers. DNA was extracted by PCI method and genotypic data was generated by PCR amplification of targeted loci. Results showed low-to-moderate level of genetic diversity in terms of number of alleles (Na), allelic richness (Ar), effective number of alleles (Nae) and heterozygosity (Ho and He). The observed heterozygosity (Ho) declined in comparison to the expected heterozygosity (He) in the wild stocks, with mean values ranging from 0.667 to 0.718 and 0.786 to 0.853, respectively. Significant deviation from H-W test was observed in 6 out of 25 tests. The population differentiation (FST) estimates were low (0.0013 to 0.0367) and insignificant. AMOVA showed that 62.38% genetic variation existed within individuals while 4.93% between populations. All of the populations were divided into two clusters by the UPGMA dendrogram constructed for genetic relatedness analysis. The study will be helpful in understanding the impact of genetic integrity on fish populations and, for sustainable management of fish genetic resources