Although phylogeographic patterns of freshwater decapods elsewhere in Australia are well documented, little is known of the phylogeography and biogeography of the endemic freshwater fauna of south-western Australia. Here, the phylogeographic structure of a freshwater crayfish, Cherax preissii Erichson, 1846, was investigated to determine contemporary and historical patterns of gene flow and to examined evolutionary and biogeographical scenarios. Allozyme and cytochrome c oxidase subunit I mitochondrial DNA data were collected from 15 populations, sampled across the known C. preissii distribution. Both markers revealed a clear distinction and separation among populations occurring in the north-western and southern portions of the distribution. Inferences of allopatric fragmentation and molecular dating attributed the divergence of the aquatic fauna of these regions to periods of Pliocene–Pleistocene aridity. Connectivity appeared to be greater within each of these regions. Evidence suggested contemporary, but not ongoing, gene flow, particularly within the southern region. This was possibly facilitated by dispersal during pluvial Pleistocene periods or drainage connectivity during episodic marine regressions. The divergence and distributions of these lineages parallels patterns seen in other freshwater crayfish of the region. More explicit investigation of these and further fine-scale phylogeographic studies may contribute to the understanding of biogeography and evolution in the south-west, and may further refine currently recognised biogeographical regions.