Chishui River, one of the first-level tributaries in the Yangtze River upstream, still maintain natural flow state and is an important breeding and protecting area for rare fish species. It is essential to understand the current status of diversity and community structure characteristics of fishes in the Chishui River for aquatic diversity conservation and ecological restoration. This study sampled 52 sites in the Chishui River in September 2021. Fish diversity was surveyed using environmental DNA metabarcoding (eDNA), with 12S rDNA Miseq high-throughput sequencing. The Shannon-Wiener index, Pielou index, and Simpson index of the upper, middle and lower reaches of the Chishui River were calculated. The dominant species of the fish community were determined by calculating occurrence frequency (fi) and dominance index (Y). Community diversity and species distribution characteristics were computed by Non-metric Multidimensional Scaling (NMDS) analysis, Adonis Permutation Multivariate Analysis of Variance (PerMANOVA) and analysis of Similarity (ANOSIM). A total of 77 fish species was found in the Chishui River, belonging to 6 orders, 18 families and 62 genera, among which 16 fish species endemic to the Yangtze River. Cypriniformes was the primary order, accounting for 87.87%. The mainly feeding habits were omnivorous and carnivorous in the Chishui River. In terms of community structure, more fish was found in the lower water environment. Zacco platypus, Sinogastromyzon sichangensis, Pseudorasbora parva, Pseudobagrus ussuriensis and Acrossocheilus yunnanensis were the dominant species in the Chishui River. The Shannon index showed significant differences between the upstream and downstream of the Chishui River (P<0.05). The fish community structure was also significantly different between the upstream and downstream communities (P<0.01). The number of fish species increased from upstream to downstream of the Chishui River. Altitude, velocity, pH, conductivity and temperature were main environmental factors in affecting fish diversity in the Chishui River. This study provides an exploratory assessment for applying eDNA technology to investigate fish diversity in the Chishui River, which will contribute to aquatic biodiversity conservation in river ecosystems.