Using environmental DNA (eDNA) metabarcoding, we compared fish diversity in two distinct water bodies within the Yangtze River Basin with known populations of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis; YFP): the Tian-e-Zhou Reserve and Poyang Lake. We aimed to create a fish surveying tool for use in the Yangtze River Basin, while also gaining a better understanding of the prey distribution and diversity within two of the remaining strongholds of YFP. 16S rRNA universal primers were developed to amplify fish eDNA. After high-throughput sequencing and stringent data filtering, we identified a total of 75 fish species (6 orders, 9 families, 57 genera) across seasons and regions. Nine of the 75 fish species were among the 28 known YFP prey species, three of which were detected in all water samples. Our eDNA metabarcoding identified many species that had been previously captured using traditional netting practices, but also numerous species not previously collected in these water bodies. Fish diversity was higher in Poyang Lake than in Tian-e-Zhou Reserve, as well as higher in the spring than in summer. These methods provide a broadly applicable tool to quantify fish diversity and distributions throughout the Yangtze River Basin, and to inform conservation strategies of YFP.