A total of 36 environmental DNA samples were collected in 12 sections of the mainstream of the Wujiang River (11 cascade hydropower reservoir areas and the Fuling section entering the Yangtze River) in July 2021 to investigate the differences in fish diversity among the reservoir areas of Wujiang’s cascade hydropower stations, and explore the impact of hydropower development on fish diversity. Meanwhile, the fish diversity and species composition structure of each river section were analyzed using environmental DNA metabarcoding technology based on CO Ⅰgene sequence. A total of 83 864 valid CO Ⅰ sequences were obtained from environmental DNA metabarcoding, and 423 OTUs were obtained after clustering by sequence similarity ≥97%, based on the alignment and annotation analysis of the self-built database, a total of 32 species of freshwater fish were detected in the entire Wujiang River, which belongs to 5 orders, 9 families and 25 genera, among which the Cypriniformes was the most species of all fish, accounting for about 56.25%. The results showed that the fish species diversity and species composition structure were some differences in the cascade hydropower reservoir area of the Wujiang River, indicating that the long-term barrier effect of cascade hydropower partially affected fish diversity and species composition structure. This study uses environmental DNA metabarcoding technology to investigate the fish diversity and species composition structure of the Wujiang River, which is helpful in understanding the current status of fish resources in the mainstream of the Wujiang River and provides certain scientific and technical support for the protection of fish diversity in the Wujiang River Basin.