1. The highly variable hydrology of dryland rivers has important implications for population dynamics in these systems. In western Queensland, fluctuations in subpopulation size are likely to lead to local bottlenecks and extinctions, increasing the need for connectivity and gene flow to maintain population viability. 2. Using microsatellite markers, we explored evidence for this metapopulation structure in two species of freshwater fish (Maquaria ambigua and Tandanus tandanus) and one crustacean (Macrobrachium australiense) in a sub-catchment of the upper Murray–Darling Basin, Australia. 3. Overall, we found very weak genetic structure for all three species. Two species (M. ambigua and M. australiense) showed some significant genetic structure that did not correlate with geographic distance. However, decomposed pairwise regression analysis revealed evidence for intense genetic drift at the waterhole scale, suggesting that local bottlenecks are driving what little genetic structure does exist for these species. 4. The results identify the local impact of bottlenecks on genetic diversity, but highlight the importance of gene flow in maintaining population viability in these highly variable systems. As the impacts of bottlenecks are likely to be tempered by gene flow, it is suggested that the maintenance of connectivity is of paramount importance in this dryland system.