Mahseer (Tor tor) is an important game fish in southern Rajasthan, India, and has recently been listed as an endangered species. The present study aimed to provide robust taxonomic classification of the species by calculating the haplotype gene frequency of Mahseer indigenous to the water bodies of southern Rajasthan. Thirty-two Tor tor samples were collected from eight sites in southern Rajasthan, India. The Cytochrome oxidase I (COI) gene was sequenced from all 32 samples. Haplotype frequencies of the samples collected were measured and phylogenetic analysis was performed. We observed that the Tor tor samples collected from the eight sites could be divided into three haplotypes, hap_1, hap_2, and hap_3. Samples collected from five sites (Chambal river, Rana Pratap Sagar, Bassi dam, Kali Sindh river, and Parwan river) exhibited the same COI haplotype, hap_1. Tor tor hap_2 was found only in samples from Badi lake; samples from Daya dam and Madar tank exhibited only Hap_3, giving a clear idea about phylogeny and group affiliations. This data can be used while repopulating the fish, which will contribute to better ecosystem management.