Freshwater biodiversity is facing a great crisis due to the looming threat of biodiversity loss. Some important target areas are difficult to survey owing to their accessibility, making them susceptible to data deficiencies. In this study, we surveyed 52 sites using environmental DNA techniques to investigate fish biodiversity in the Chishui River Basin of the Yangtze River. A total of 96,031 valid fish sequences were read, resulting in the identification of 77 species belonging to six orders, 62 genera, and 18 families. The dominant orders were Cypriniformes, Siluriformes, and Perciformes. Among the identified fishes, 71 were native and six were exotic, with the native fishes including 16 endemic fishes from the upper reaches of the Yangtze River. The upstream and downstream Shannon-Wiener indices differed significantly, the Shannon-Wiener and richness indices of the tributaries in the upstream section were significantly higher than those of the tributaries in the downstream section, and we found that the Datong River is the most diverse secondary tributary of the Chishui River. Among the environmental factors in the Chishui River Basin, altitude and electrical conductivity had the greatest influence on fish diversity (P <0.01), whereas human factors had little effect. Our findings highlight the application of environmental DNA technology to modern biodiversity surveys and illustrate that the Chishui River Basin is primarily affected by environmental factors at this stage. However, continuing efforts are needed to protect freshwater biodiversity, and additional research is required to better understand the complex interplay between human activity and environmental factors.