Dryland rivers are globally widespread and regionally important, and understanding their ecology is critical for sustaining ecosystem processes and biodiversity. The dry tropics are characterized by episodic summer rainfall. Most of annual river flow occurs in a short period of time, after which rivers typically contract into a series of waterholes of varying permanence and hydrologic connectivity. We investigated how seasonal environmental factors affected macroinvertebrate assemblage composition in dry-tropics rivers at the river, site, and habitat scales. We assessed biophysical characteristics, including water physicochemistry, riparian-zone condition, and macroinvertebrate assemblage composition at 15 sites on 4 unimpounded rivers in the Burdekin catchment, north Queensland, Australia. We used permutational analysis of variance and multiresponse permutation procedures to identify differences between a priori groups, and illustrated the results with principle components analysis (biophysical data) and nonmetric multidimensional scaling (invertebrate assemblage data). Biophysical variables were spatially and temporally variable, and all rivers and most sites differed significantly. Macroinvertebrate assemblages also differed significantly among rivers, sites within rivers, habitats, and seasons. Assemblages from the same habitat but different sites were more similar than assemblages from different habitats within the same site. Thus, the dynamic environment of dryland river systems drives naturally dynamic and variable macroinvertebrate assemblages across a range of scales. The differences among sites from the same river were of particular importance from a monitoring standpoint because they indicated that extrapolating whole-river condition from a few sampled sites would be difficult.