Reservoir impoundment affects fish habitat condition, in turn likely influencing the spatial and temporal distribution patterns of fish assemblage structure in the impounded area. Fishing activity and instream gravel and sand mining are important anthropogenic factors, and flow discharge, water temperature, and precipitation are important natural environmental variables; both anthropogenic and natural factors likely have impacted fish assemblage structure in impounded and unimpounded waterbodies. We used multivariate analysis to evaluate the combined effects of two adjacent reservoir impoundments in the main stem of a large river (Jinsha River; the upper segments of the Yangtze River), overfishing, instream gravel and sand mining, the natural flow regime, and other environmental variables (water temperature and precipitation) on fish assemblage structure in the Heishui River, a small, free‐flowing tributary upstream of the mainstream reservoirs. Fish assemblage data and abiotic data collected over 10 years (2008, 2010, and 2011–2017) were analyzed. The results showed that fish assemblages of the Heishui River temporally clustered into two groups that were strongly associated with the two downstream cascade reservoirs (Xiangjiaba and Xiluodu reservoirs) in the main‐stem Jinsha River. The relationships between fish assemblage structure and abiotic factors were tested using a distance‐based linear model and were visualized using distance‐based redundancy analysis, showing that the main factors explaining the interannual and seasonal variation in fish assemblage structure were instream gravel and sand mining, mainstream reservoir impoundment, and seasonal flow regime. Among these, instream gravel and sand mining in this tributary played the most significant role in affecting the temporal variation of fish assemblage structure and explained 34.12% of the total variability. Mainstream reservoir impoundment and seasonal flow regime exerted secondary effects on the temporal variation of fish assemblage structure and together explained 13.39% (7.00% and 6.39%, respectively) of the total variability. This study suggests that the relevant administrative agencies should focus more on free‐flowing habitat maintenance in this tributary and the control of instream gravel and sand mining.