The influence of weirs on the distribution of freshwater mussels was investigated in the Hawkesbury-Nepean River, Australia. Distribution of species and densities of size classes were strongly correlated with catchment level factors (e.g. location around a major impoundment, stream order). At catchment scale, weir height, presence of a fish barrier, fish ladder type and position above or below small weirs did not influence the presence/absence of mussel populations. Lower mussel densities in the upper catchment may therefore reflect inhibition of host fish migration. Where present, weir height and geomorphic reach type were linked to differences in densities among species. Geomorphic reach-based differences were reflected by the Hyridella species, but not Velesunio ambiguus. When population structure was described by size class distribution, there were significant differences between densities of small and medium mussels from weirs above, compared to weirs below, a major impoundment, but not for large mussels. Upstream populations may therefore be functionally extinct. Distribution of mussel size classes differed among geomorphic reach types with highest densities for each class found in the least human-impacted reaches. Small mussels were almost invariably found below the major impoundment, most frequently below weirs. Distribution patterns were inconsistent across species, suggesting habitat preference. V. ambiguus and Hyridella australis were most abundant in shale reaches, where assemblages were influenced by fish ladder type. Hyridella depressa and H. australis dominated in sandstone gorges and straights with assemblage density related to weir height. In upper catchment sandstone reaches, mussel assemblages comprising predominantly V. ambiguus were influenced by fish ladder type and weir height. While multiple factors defined localized distribution, large impoundments were linked with reduced population densities. The probable mechanism is the restriction of host fish movement and resulting lack of recruitment. In the Hawkesbury-Nepean River, smaller weirs also seriously impacted recruitment.