Species are considered as a fundamental unit of biodiversity. Therefore, the prerequisite for biodiversity management and conservation is to know the number of species one is dealing with. Consequently, the need of the present study was conceptualized, which dealt with the comprehensive molecular appraisal of Kaladan's Fish fauna. A total of 291 specimens representing 49 species, 28 genera, 11 families, and 4 orders, were collected from 11 sampling stations situated along the main Kaladan River and its four major tributaries, i.e. Tiau, Tuipui, Mat, and Tuichang, flowing in Mizoram state of India (part of Indo-Myanmar biodiversity hotspot) and COI sequences of all the 291 samples were generated. All the analyses conducted in the present study, i.e. K2P genetic divergence, bPTP and Neighbour-Joining suggest that DNA Barcoding is an efficient and reliable tool for species identification and deciding the species boundary. Most of the species of Kaladan showed the clear existence of barcode gap. However, the presence of intra-specific and inter-specific genetic distance overlap in two species revealed the existence of putative sibling species or hidden taxa. This study also revealed the presence of two cryptic species and putative previously unknown species of genus Garra and Schistura. The COI barcode database of Kaladan's fish fauna, established in the present study, may serve as a reference library for accurate identification of fishes and will help ichthyologist, researcher, students, biodiversity managers and policy makers in proper planning with regard to conservation and management of the resources.