Humans have exaggerated natural habitat fragmentation, negatively impacting species dispersal and reducing population connectivity. Habitat fragmentation can be especially detrimental in freshwater populations, whose dispersal is already constrained by the river network structure. Aquatic insects, for instance, are generally limited to two primary modes of dispersal: downstream drift in the aquatic juvenile life stages and flight during the terrestrial winged adult stage. Yet the impacts of large hydropower dams can make rivers uninhabitable for incoming (drifting) juvenile insects, with remaining refugia found only in tributaries. The ability of adult aquatic insects to traverse such river stretches in search of suitable tributary habitat likely depends on factors such as species-specific dispersal ability and distance between tributaries. To explore the intersection of natural and human-induced habitat fragmentation on aquatic insect dispersal ability, we quantified population genetics of three taxa with varying dispersal abilities, a caddisfly (Hydropsychidae, Hydropsyche oslari), a mayfly (Baetidae: Fallceon quilleri), and a water strider (Veliidae: Rhagovelia distincta), throughout tributaries of the Colorado River in the Grand Canyon, Arizona, USA. Using 2bRAD reduced genome sequencing and landscape genetics analyses, we revealed a strong pattern of isolation by distance among mayfly populations. This contrasts with caddisfly and water strider populations, which were largely panmictic. Analysis of thousands of informative single nucleotide polymorphisms showed that realized dispersal ability may not be accurately predicted by species traits for these widespread species. Principal components analysis revealed a strong division between caddisfly populations upstream and downstream of Havasu Creek (279 km through the 390 km study reach), suggesting that the geography of the Grand Canyon imposes a dispersal barrier for this species. Our use of genetic tools in the Grand Canyon to understand population structure has enabled us to elucidate dispersal barriers for aquatic insects. Ultimately, these data may be useful in informing effective conservation management plans for understudied organisms of conservation interest.