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To investigate the longitudinal gradient patterns and the factors influencing fish taxonomic and functional groups, a survey involving 28 sampling sites was conducted in the Anning River in November 2020 and April 2021. A total of 60 fish species were collected, and these species were classified into 4 locomotor groups and 4 trophic groups, resulting in 11 combined functional groups based on locomotor and trophic characteristics. The results of two-way cross similarity analysis (ANOSIM) revealed significant changes in both the fish taxonomic and functional groups along the longitudinal gradient, while no significant seasonal variation was observed in either group. Spatial changes in taxonomic and functional groups along the river were primarily due to turnover processes, as indicated by the similarity percentage analysis. Variance partitioning analysis demonstrated that local habitat conditions and land use/cover explained 37.56% and 41.33% of the spatial variation in taxonomic and functional organizations, respectively. Notably, the influence of landscape features on taxonomic organization was less significant than that of local habitat variables. Nevertheless, landscape conditions played a more important role than local environmental factors in explaining variations associated with functional organization. Our study helps in understanding the spatial patterns and formation mechanisms of taxonomic and functional groups driven by multiple-scale environmental factors in mountain rivers. In addition, our results showed that the response of functional groups to different scale environmental factors differ from taxonomic groups.