AbstractFreshwater ecosystems are under great threat from humans, among which habitat heterogeneity is the most obvious, being one of the important reasons for the decline of fish diversity. This phenomenon is particularly prominent in the Wujiang River, where the continuous rapids of the mainstream have been divided into 12 mutually isolated sections by 11 cascade hydropower reservoirs. Based on the fact that conventional survey methods are more harmful to the ecological environment, the efficient and noninvasive environmental DNA metabarcoding (eDNA) approach was used in this study to conduct an aquatic ecological survey of the 12 river sections of the mainstream of the Wujiang River. A total of 2299 operational taxonomic units (OTUs) were obtained, corresponding to 97 species, including four nationally protected fish species and 12 alien species. The results indicate that the fish community structure of the Wujiang River mainstream, which was originally dominated by rheophilic fish species, has been changed. And there are differences in fish species diversity and species composition among the reservoir areas of the mainstream of the Wujiang River. The fish species in the area have gradually declined under the influence of anthropogenic factors such as terraced hydropower and overfishing. The fish populations consequently have demonstrated a tendency to be species miniaturized, and the indigenous fish are severely threatened. In addition, the fish composition monitored by the eDNA approach was found to be close to the fish composition of historical information on the Wujiang River, indicating that eDNA approach may be used as a complementary tool to conventional methods in this basin.