This study of Genetic Differentiation among Himalayan & local Mahseer concerns with a conservation plan is a fundamental part of the hydro projects still in the pipeline. In-situ conservation and gene banking also serve as the best medium for the same. Despite conservation programs, re-examination and revision of these conservation areas to update the number and lists of endangered status are mandatory. Fish samples collected from 8 different waterbodies {Chambal River, Rana Pratap Sagar Dam, Bassi Dam, Kali Sindh River, Parwan River, Badi Lake, Daya Dam, and Madar Tank} representing two major drainage systems (the Bay of Bengal and the Arabian Sea) of India. For the cytochrome oxidase subunit I, a total of 32 sequences was generated, whereas, for the ATPase 6 gene, a total of 66 sequences were generated. Two mitochondrial regions COI and ATPase6 were amplified using the set (Forward & Reverse primer set) of published primers (COI-Fish F-1/Fish R-1. BOLD-IDS was used to test the efficiency of DNA barcoding as a species identification tool using a blind sampling test, the sample selected & sequenced were known only to the submitting individual. Mismatch distributions to evaluate the hypothesis of recent population growth with 99,999 permutations as implemented in ARLEQUIN 3.0.1 was used. This distribution is unimodal in populations that have passed through a recent demographic expansion and are multimodal in stable populations. A DNA sequence of Himalayan Mahseer (Tor tor) was downloaded from NCBI Gene Bank and the sequences generated in the present study were compared (Using MEGA7 Software) with the downloaded sequence. It's derived that the stocks of Rajasthan Mahseer are quite distinct from the Himalayan stocks. The minimum genetic (116.06) distance was noticed for both Daya dam and Madar Tank and the highest (125.35) was for Bassi Dam. The evolutionary history was inferred using the Minimum Evolution method. The optimal tree with the sum of branch length 123.79668059. The significantly higher genetic distance between Rajasthan and Himalayan stocks could be due to geographic location and mainly isolated aquatic resources. Similar findings have also been reported by earlier workers.