Ompok bimaculatus commonly called as Indian butter catfish is a commercially important food fish and distributed throughout India and Southeast Asia. Genetic differentiation of O. bimaculatus was investigated using cytochrome b (cyt b) gene of three different riverine populations. A total of 37 sequences of cyt b gene (1118 bp) were generated from O. bimaculatus. The mitochondrial DNA analysis revealed that O. bimaculatus has relatively high genetic diversity. The estimated haplotype and nucleotide diversity were 0.063 and 0.005, respectively. The analysis of molecular variance indicated 73.53% genetic variability between the populations and 26.47% genetic variation within population. The understanding of genetic differentiation of O. bimaculatus populations will play a key role in conservation and management of the important endangered fish species.