Arunachal Pradesh, the largest state of North-East India covers almost 60.93% of the Eastern Himalayan hotspot. Fish diversity and species identification is utmost important for fisheries management. But, in some cases morphological characteristics based identification is difficult for a non-specialist to perform. In view of the above, the present study emphasized on the assessment of DNA barcoding, phylogenetics and genetic diversity of fish species in the Ranganadi River, Arunachal Pradesh, India. India. Arunachal Pradesh, the largest state of North-East India covers almost 60.93% of the Eastern Himalayan hotspot. Altogether 114 specimens, representing 22 species, belonging to 3 orders and 5 families were successfully barcoded and found to be 98-100% identical from both GenBank and BOLD databases. Out of these 22 fish species, it was found that one species assessed was Endangered, three species as Near Threatened and one species as Vulnerable. A Neighbour Joining (NJ) tree was constructed using Rstudio for the purpose of a phylogenetic analysis of the identified species. The barcoding gap analysis using K2P, P-distance and Jukes-Cantor was done to detect the presence of cryptic species and barcoding success. The nucleotide base composition and genetic distance analysis were also performed, using MEGA 6.0. DNA Sequence Polymorphism v6.12.03 analysis revealed the nucleotide diversity (p) and haplotype diversity (Hd). The Hd for the whole dataset was found to be 0.975, which showed high genetic diversity in the Ranganadi River. Both morphological key identifying characters and molecular data corroborated the phylogenetic analysis. This COI barcode library, generated in the present study, not only helped in species identification and molecular study, but also in cryptic species identification.