To develop an effective conservation and management strategy, it is required to assess the biodiversity status of an ecosystem, especially when we deal with Indo-Myanmar biodiversity hotspot. Importance of this reaches to an entirely different level as the hotspot represents the area of high endemism which is under continuous threat. Therefore, the need of the present study was conceptualized, dealing with molecular assessment of the fish fauna of Indo-Myanmar region, which covers the Indian states namely, Manipur, Meghalaya, Mizoram, and Nagaland. A total of 363 specimens, representing 109 species were collected and barcoded from the different rivers and their tributaries of the region. The analyses performed in the present study, i.e. Kimura 2-Parameter genetic divergence, Neighbor-Joining, Automated Barcode Gap Discovery and Bayesian Poisson Tree Processes suggest that DNA barcoding is an efficient and reliable tool for species identification. Most of the species were clearly delineated. However, presence of intra-specific and inter-specific genetic distance overlap in few species, revealed the existence of putative cryptic species. A reliable DNA barcode reference library, established in our study provides an adequate knowledge base to the groups of non-taxonomists, researchers, biodiversity managers and policy makers in sketching effective conservation measures for this ecosystem.