Pengba fish, Osteobrama belangeri is a freshwater inhabitant, highly endemic, threatened and economically important minor carp for its food value. In the present investigation, population structure of O. belangeri was examined using mitochondrial ATPase 6/8 gene from geographically distinct locations along the Indo-Burma biodiversity hotspot. A total of 80 individuals were collected belonging to natural and farm populations. The hierarchical analysis of molecular variance and conventional Fst values (0.825 in ATPase 6/8, p < .05) indicated significant genetic structure among populations. The result showed that ATPase 6/8 genes are potential marker in determining the genetic divergence between natural and farm populations of O. belangeri from North-East India.