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India’s third longest river, the Narmada, is studied here for the potential effects on native fish populations of river fragmentation due to various barriers including dams and a waterfall. The species we studied include a cyprinid fish, Catla catla, and a mastacembelid, Mastacembelus armatus, both of which are found in the Narmada. Our goal was to use DNA sequence information from the D-loop region of the mitochondrial DNA to explore how this fragmentation could impact the genetic structure of these fish populations. Our results clearly show that these barriers can contribute to the fragmentation of the genetic structure of these fish communities, Furthermore, these barriers enhance the effects of natural isolation by distance and the asymmetry of dispersal flows. This may be a slow process, but it can create significant isolation and result in genetic disparity. In particular, populations furthest upstream having low migration rates could be even more subject to genetic impoverishment. This study serves as a first report of its kind for a river system on the Indian subcontinent. The results of this study also emphasize the need for appropriate attention towards the creation of fish passages across the dams and weirs that could help in maintaining biodiversity. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-385) contains supplementary material, which is available to authorized users.