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Hydrological connectivity in river systems facilitates the movement of animals across riverine landscapes and influences fish habitat but is threatened by climate change and water resource developments. We studied fish assemblages across a large wet–dry tropical river system in northern Australia that is under consideration for new agricultural and water resource developments, which are expected to alter hydrological connectivity. We explored relationships between the environment and several biotic variables; fish taxonomic composition, species turnover, and species trait presence, quantifying how they were related to hydrological connectivity. Environmental dissimilarity of sites was influenced by hydrological connectivity variables, including flow, elevation, and river distance. Environment characteristics and hydrological connectivity together were important predictors of fish taxonomic composition. Fish species turnover was highest in headwater sites, and species presence absence was related to feeding and reproductive traits. Our results suggest that habitat specialists and species with reproductive traits that depend on hydrological connectivity, such as diadromous species, are most vulnerable to declines in relative abundance following a reduction in connectivity, which would lead to range contractions within catchments. Maintaining habitats that support taxonomically and functionally unique fish assemblages, such as wetlands and headwater streams, is important for maintaining biodiversity. [ABSTRACT FROM AUTHOR]