Abstract: The Chishui River is a first-level tributary of the upper reaches of the Yangtze River with complex and diverse habitat types, and plays an important role in the protection of rare and endemic fish resources in the upper reaches of the Yangtze River. In order to understand the health level of the Chishui River Basin, a fish survey was conducted at 31 sites on the main stream and primary tributaries of the Chishui River in May and September 2020. A total of 6288 fish species belonging to 4 orders, 10 families, 26 genera and 90 species were collected. The Shannon-Wiener index ranged from 0.21 to 2.87 with an average value of 1.48±0.46. The Simpson and Pielou index ranged from 0.08 to 0.91 with average values of 0.69±0.14 and 0.13-0.97 with an average value of 0.76±0.13, showing a gradual increase trend from the upstream to downstream. The F-IBI index was used to evaluate the health status of the Chishui River, the core index system included 10 indicators including the total number of species, the number of cypriniforms, the number of Gobioninae, the number of Culterinae, etc. The F-IBI value of each sampling point was calculated by the ratio method, and five health grades were classified into ‘healthy (F-IBI≥5.99)’, ‘subhealthy (4.49≤F-IBI<5.99)’, ‘average (2.99≤F-IBI<4.49)’, ‘poor (1.50≤F-IBI<2.99)’ and ‘extremely poor (F-IBI<1.50)’ to evaluate the health status of each sampling point. In general, the surveyed sites were in a healthy state, among which 20 sites were in healthy state, 5 sites were in a sub-healthy state, 3 sites were in a general state, and 3 sites were in a poor state. The F-IBI values gradually increased from upstream to downstream, and there was a significant difference between the upper, middle and downstream (P<0.05). The main stream exhibited better health status than the tributaries, the poor health sites are located in the Tongzi River and the Wuma River, while the general health sites are located in the Xishui River Basin. Cypriniforms and Cyprinidae were the dominant fish fauna in the Chishui River, indicating that the F-IBI evaluation system was a good predictor of human disturbance and better reflected the health status of aquatic ecosystem in the Chishui River, which can be used as a tool for aquatic ecosystem health evaluation and resource management for the Chishui River.