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Conservation of freshwater biodiversity is a major challenge, globally. It is obvious that for management and policy making, taxonomic data and their current status become inevitable. Dudhkoshi River is one of the major tributaries of the Koshi basin, and the least explored river regarding fish diversity. In this study, we explored the fish diversity status of this river incorporating species composition, diversity indices, and (relative) abundance, and also endeavored to reveal the conservation status of its ichthyofauna for the first time. Fish sampling was carried out using combinations of fishing gears and with the assistance of local fishermen. A total of 22 species belonging to 13 genera, eight families, and three orders, making a total of 1,265 individuals were collected. Cypriniformes and Cyprinidae were recorded as the dominant order and family which accounted for 77.27% and 36.36%, respectively. Species like Schizothorax richardsonii, S. progastus, Labeo dyocheilus, Barilius bendelisis, B. barila, Glyptothorax pectinopterus and Psilorhynchus pseudecheneis were found as the most abundant species, from highest to lowest, and Botia dario, Channa barca, Eutropiichthys murius, Garra annandalei, Puntius sophore and Schistura multifasciata were recorded as the occasional (rare) species. The Shannon-Wiener’s diversity index shows that the overall fish diversity status of Dudhkoshi River was medium. While the majority of species belong to the Least Concerned (LC) category accounting for 67%, the four species, Neolissochilus hexagonolepis, Psilorhynchus pseudecheneis, Schizothorax progastus and S. richardsonii belong to NRDB status; those species had been proposed for legal protection in Nepal. To appreciate more generalized inferences about the fish diversity status of Dudhkoshi River, a comprehensive study integrating more seasons (e.g. winter and rainy) is highly recommended for future study.