DNA barcode assisted Molecular Taxonomy for species identification aided us to employee this technique in discrimination of fishes from Narmada river basin, one of the longest river of India. During our study we found high genetic diversity between individuals isolated by barriers (dams/waterfalls) this facilities us to study, how barriers contribute to the fragmentation of the genetic structure in the fish communities. This study serves as first report of its kind for river system on the Indian subcontinent.