Using genetic techniques (mtDNA, microsatellites and allozymes), this study investigated patterns of gene flow in two plotosid catfish species (Neosilurus hyrtlii and Porochilus argenteus), at various hierarchical scales in the dryland rivers of western Queensland, Australia. The study area constituted two major catchments, Cooper and Darling, representing arid and semiarid systems, respectively. Results generally conformed to expectations, with high levels of gene flow observed within catchments and limited contemporary gene flow evident across catchment boundaries. However, the isolation between catchments was more recent than expected, occurring approximately 40 000–72 000 years ago. Also contrary to predictions, genetic structure within the Cooper catchment did not fit the stream hierarchy model of genetic differentiation, which there was evidence of in the Darling catchment. This was hypothesized to relate to the different climatic regimes and hydrological inputs in each system, leading to a more genetically homogeneous system in the Cooper than in the Darling system. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 457–467.