The ecological effects of small run-of-river dams on aquatic ecosystems are poorly understood, especially on downstream benthic algal communities. We examined impacts of such dams on the benthic diatom community at a regional scale in the Xiangxi River, China. A total of 90 sites were visited, which were divided into five habitats (H1–H5) according to impact extent of each dam. Using partial least squares (PLS) modeling, we developed two predictive models (diatom species richness and total diatom density) based on environmental variables of an unregulated habitat (H1). These models were then used to predict species richness and total densities at impacted habitats (H2–H5) and residuals, i.e. the differences between observed and predicted values, were used to evaluate impact strength of flow regulation. Significant impacts of flow regulation on diatom species richness were detected at three impacted habitats (H3–H5), where observed species richness were significantly higher—70.6, 63.9 and 46.6%, respectively—than predicted values. Then, possible mechanisms for observed impacts were discussed. Further research is necessary to address the potential negative impacts of cascade run-of-river dams on other aquatic organisms in different seasons, and to explore more appropriate mechanisms for such impacts, which may lead to sustainable management strategies and help to determine the optimal ecological water requirement for the Xiangxi River.