The freshwater isopod genus Amphisopus is one of only two phreatoicidean genera in Western Australia with wide distributions and containing multiple described species. Two species (Amphisopus annectans and A. lintoni) are known from the south-western part of the state. With recent sampling extending the known range of Amphisopus and the recorded possibility of an undescribed species, this study aimed to examine genetic species boundaries and to detect additional species diversity. Isopods were sampled from across the range, and genetic structure was examined using mtDNA sequence data from a COI fragment and data from ten polymorphic allozyme loci. While allozyme data supported the clear separation of the known species, phylogenetic analyses presented three divergent monophyletic, geographically restricted clades occurring in the western, eastern and central parts of the Amphisopus distribution. These corresponded to A. annectans, A. lintoni and an undescribed species, respectively. The latter’s status was supported by its phylogenetic position relative to the known species and by the extent of differentiation observed among other phreatoicidean taxa. As with other taxa from the region, divergence times among these three species supported aridity in the Miocene–Pliocene as a driver of diversification, and suggested the existence of wetter refugial areas in south-western Australia.