The Qilian Mountain Basin, on the north-eastern edge of the Qinghai-Tibet Plateau (QTP), supports a high diversity of native and endemic fish. However, the detailed species inventory and distribution patterns concerning fish in the whole Basin remain unknown, which hinders the conservation of biodiversity and assessment of ecological health. We compiled a comprehensive species richness and distribution database of freshwater fish in the Qilian Mountain Basin, based on field investigations and exhaustive data collection from 50 rivers or lakes. Then, we elucidated a distribution pattern using clustering and ordination analyses based on a βdissimmatrix with species presence/absence data. A total of 79 freshwater fish species within eight orders, 17 families and 42 genera were recorded. The Qilian Mountain Basin could be grouped into six systems, which match the six Basins (i.e. Heihe River Basin, HHR; Qaidam Basin, QDM; Qinghai Lake Basin, QHL; Shule River Basin, SLR; Shiyang River Basin, SYR; Yellow River Basin, YR), based on the fish distribution pattern. Additionally, the spatial pattern of species distribution showed the distance decay of taxonomic similarity. Our results demonstrate that riverine connectivity resulting from historical processes plays a vital role in shaping the freshwater ichthyofauna of High Central Asia. These findings will be valuable for future systematic conservation of fish in the Qilian Mountain Basin.