This study examined the phylogenetic relationship and identification of five Mahseer species (Tor putitoro, Tor tor, Tor khudree, Tor chelynoides and Neolissochilus hexogonolopis) using partial sequencing of a Cytochrome Oxidase I (COl) DNA barcodes. The sequence analysis data showed that 134 (21.61%) sites out of 628 sites were variable without insertion or deletion. Rate of transition (70.5%) were higher than transversion (29.41%). There was a high inter-species divergence (range 4.1% to 12.2%) in Mahseer species as compared to intra-specific sequence divergence (1.7% for T. putitora, 1.2% for T tor, 1.4% for T. khudree, 3.0% for T chelynoides, 0.26 % for N. hexagonolopis). The phylogenetic tree, constructed by maximum parsimony, maximum likelihood and unweighted pair group average methods revealed similar results suggesting that T. putitoro, T. tor and T. khudree had a close relationship to each other while maximum divergence was observed in T. chelynoides, which was also confirmed by the genetic distance data. The results indicate that COl sequencing or bar-coding is useful in unravelling phylogenetic relationship and identification of Mahseer species.