Understanding the patterns and ecological determinants of β diversity is critical for community ecology and conservation biology. However, only few studies have investigated fish β diversity in large rivers, and even fewer have included multiple facets of biotic and abiotic diversity indices simultaneously. Based on sampling data from the Chishui River basin in 2019 and 2020, we calculated fish species, functional, taxonomic, and environmental β diversity, as well as the relative contribution of individual sampling site to each facet of β diversity (also known as site uniqueness). Further, we assessed the degree of complementarity and redundancy between the calculated indices of site uniqueness. By doing these, we tried to provide a scientific basis for the conservation and management of fish resources in the Chishui River basin, which is a pioneer area for the protection of the upper Yangtze River. The results showed that a total of 125 fish (sub) species belonging to 7 orders, 20 families and 80 genera were collected from the Chishui River basin, including 112 native fish (sub) species and 13 exotic (sub) species. Species β diversity of fish assemblages were 0.719, and species uniqueness of sampling sites S1, S2, S22 and S27 was significantly different from other sites. Functional β diversity of fish assemblages were 0.009, with sampling sites S10, S20, S21, S28 and S36 having significantly different functional uniqueness values compared with other sites. Taxonomic β diversity of fish assemblages were 0.004, with sample sites S20, S21, S28 and S32 having significantly higher taxonomic uniqueness in comparison with other sites. Environmental uniqueness of sampling sites ranged from 0.010 to 0.058, and the four highest sites were S22, S29, S33 and S34. Fish species uniqueness of sampling sites showed a significant U-shaped relationship with fish richness, that is, species uniqueness of sampling sites firstly decreased and then increased with fish richness. There was a high degree of complementarity between different facets of diversity indices, and functional and taxonomic uniqueness of sampling sites explained more diversity variation than species uniqueness of sampling sites. This study suggests that fish diversity assessments need to integrate different facets of biotic and abiotic diversity indices, as they can be useful in revealing spatial and temporal variation of freshwater fish community structure and the underlying mechanisms.