The Zhougong River is one of the most important tributaries of the Qingyi River in upper Yangtze River drainage, Sichuan Province, southwest China. The river has been dammed in several places by hydroelectric stations and thus, we aimed to characterize the longitudinal and seasonal patterns of fish assemblage structure in it and whether anthropogenic perturbations have impacted fish assemblages. An extensive survey of fish was conducted in spring (April), summer (August) and autumn (November) of 2015, and winter (February) of 2016 at a total of 18 sampling locations across the river's watershed. It was found that fish abundance, richness and Shannon-Wiener diversity all increased from upstream to downstream. Fish assemblage showed significant spatial differences between the upstream, midstream and downstream, while there were no differences between seasons as was expected. The spatial variation in the abundance of Rhodeus sinensis, Schizothorax prenanti, Pseudorasbora parva, Zacco platypus and Abbottina obtusirostris were considered to contribute most to the spatial pattern in fish assemblage. The canonical correspondence analysis (CCA) results revealed that spatial variations of fish community in the Zhougonng River were significantly correlated with water velocity, total phosphorus and water depth. Our findings indicate that spatial differences and no seasonal change in fish assemblage were partially attributed to the physicochemical changes caused by cascade hydroelectric dams (e.g., channel blocking, hydrological alterations) and water pollution (e.g., phosphorus) and it is also likely that overfishing has exacerbated impacts. We recommend that pollution prevention, dam removal, modifying dam ecological operations and fishery control may effectively lessen the impacts on fish biodiversity in the Zhougong River.