We explored fish fauna Ganga and Yamuna river in Uttarkashi district of Uttarakhand, to establish the molecular taxonomy database. A total of 133 samples were collected from various sampling sites along the entire stretch of rivers in district Uttarkashi. A region of cytochrome c-oxidase unit I (COI) gene (655 bp) was amplified using PCR and sequenced (DNA Barcoded). All the sequences have been uploaded into the NCBI GenBank (accession number assigned). Analysis of data generated showed that these 133 samples belonged to 22 species of 4 orders, 14 genera and 8 families. The genetic variability (K2P distance) distribution analysis was also carried out. The average mean distance of 22 species is 0.219 with 0.014 standard error. The mean genetic distance between 22 species ranged from 0.010%-0.362% while the mean genetic distance within the species ranged from 0.0006%-0.0048%. The lowest pairwise genetic distance observed in Schizothorax sinuatus and Schizothorax progastus i.e. 0.010% with 0.003 standard error indicating a closer phylogenetic relationship between Schizothorax sinuatus and Schizothorax progastus than other species which was confirmed by the genetic distance data. Maximum divergence were observed between Danio devario and Channa gachua i.e. 0.362% with 0.028 standard error. The maximum sequence divergence within the species is observed in Barilius bendelisis with 0.0048% while minimum sequence divergence is observed in Tor putitora with 0.0006 of cyprinidae family. Our data suggests that there is high inter-specific sequence divergence as compared to intra-specific sequence divergence and also conclude that COI sequencing (barcoding) was found to be suitable for the identification of fresh water fish species.