Dam impoundment can cause many adverse impacts on fish assemblages upstream of dams. Here, we investigated fish diversity in one plateau riverine reservoir (Wanfeng Reservoir) using environmental DNA (eDNA) metabarcoding technology. The following conclusions were drawn: (1) 39 species of fish were monitored belonging to 9 orders and 13 families in the Wanfeng Reservoir, most of which were Cypriniformes and included a variety of common farmed fish belonging to Culter, Oreochromis, Acipenser, and Clarias; (2) the fish assemblage structures in the Up (upstream section), Mid (midstream section), and RA (reservoir area section) of this reservoir was significantly different (p-value < 0.01); (3) among the 7 environmental variables that were measured (Tem, TDS, Sal, pH, DO, NH4+, and Tra), DO (p-value < 0.01) and pH (p-value < 0.05) were the main environmental stressors causing differences in fish assemblages in different sites of the Wanfeng Reservoir. This study concluded that dam construction in the karst region provided habitats for the establishment and dispersal of exotic fish.