River impoundment constitutes one of the most important anthropogenic impacts on the World’s rivers. An increasing number of studies have tried to quantify the effects of river impoundment on riverine ecosystems over the past two decades, often focusing on the effects of individual large reservoirs. This study is one of the first to use a large-scale, multi-year diatom dataset from a routine biomonitoring network to analyse sample sites downstream of a large number of water supply reservoirs (n = 77) and to compare them with paired unregulated control sites. We analysed benthic diatom assemblage structure and a set of derived indices, including ecological guilds, in tandem with multiple spatio-temporal variables to disclose patterns of ecological responses to reservoirs beyond the site-specific scale. Diatom assemblage structure at sites downstream of water supply reservoirs was significantly different to control sites, with the effect being most evident at the regional scale. We found that regional influences were important drivers of differences in assemblage structure at the national scale, although this effect was weaker at downstream sites, indicating the homogenising effect of river impoundment on diatom assemblages. Sites downstream of reservoirs typically exhibited a higher taxonomic richness, with the strongest increases found within the motile guild. In addition, Trophic Diatom Index (TDI) values were typically higher at downstream sites. Water quality gradients appeared to be an important driver of diatom assemblages, but the influence of other abiotic factors could not be ruled out and should be investigated further. Our results demonstrate the value of diatom assemblage data from national-scale biomonitoring networks to detect the effects of water supply reservoirs on instream communities at large spatial scales. This information may assist water resource managers with the future implementation of mitigation measures such as setting environmental flow targets.