The landscape of the Pilbara region of Western Australia has been relatively unchanged for 100 million years. The ancient river systems of this region might be expected to be sources of isolation and divergence for aquatic species. Hence, the occurrence of widespread groundwater taxa in this landscape offers the opportunity to examine associations between genetic diversity and drainage patterns. Pilbarus and Chydaekata are two widespread genera of subterranean amphipods endemic to the Pilbara, each occupying multiple tributaries. We used molecular data to examine the roles of drainage patterns in structuring genetic diversity. Gene flow within a tributary may be facilitated by the occasional occurrence of these amphipods in springs, which results in their downstream dispersal during episodic flooding. However, tributary boundaries may form hydrological barriers to gene flow, resulting in localised isolation of populations and divergence. Samples of both genera, collected throughout three river basins, were examined for sequence divergence in the cytochrome c oxidase I mitochondrial gene. There was no evidence of contemporary gene flow among populations of either genus, and each tributary contained highly divergent lineages, which were not associated with similar morphological differentiation. This suggests cryptic speciation has occurred, and similar phylogenetic signals in both taxa imply similar evolutionary histories. Surface populations may have been driven into subterranean refugia by the cessation of flow in the rivers, associated with Tertiary climate change, while morphological evolution may have been constrained by stabilising selection. The lack of congruence between molecular diversity and morphology raises important practical issues for conservation.