Freshwater fishery resources in Chinese rivers have been markedly impaired as the results of overfishing, damming, and watershed development. However, little is known about trophic ecology of fish assemblages in the rivers of China. In this study, fishes were collected from an upstream, midstream, and downstream site of the Moyangjiang River, Southern China, to investigate trophic niche structure using stable isotope analysis. We calculated several trophic niche metrics using δ13C and δ15N ratios of the fish assemblages at each study site. Results showed no significant differences in the δ13C ratios between the pelagic and benthic fish assemblages at each study site. This suggests a homogenous dietary base shared by all fish at each site, likely as the result of continuous water column mixing in this shallow and fast‐moving river. The upstream fish assemblage had the greatest species abundance and trophic diversity. The fish assemblage at the midstream site displayed the smallest trophic niche space (total area of the δ13C and δ15N bi‐plot space) and trophic diversity. The midstream site also showed the most depleted 13C, suggesting a more degraded habitat compared with the other two study sites. The fish assemblages at the downstream site displayed the greatest basal resource diversity, largest CR, and trophic niche space and trophic diversity. However, the fish assemblage at the downstream site also displayed the lowest species abundance, redundancy, and evenness of trophic spacing. These findings suggest that the trophic niche of the fish assemblage at the downstream site, which received resources from upstream flow, experienced greater watershed development and greater in situ production, demonstrating positive and negative impacts by anthropogenic activities. Future studies should also gather information on fish production, watershed development, and water quality, to aid in the interpretation of the stable isotope analysis of fish trophic niches.