The arid Yilgarn region of Western Australia contains numerous isolated calcrete aquifers, within which a diverse subterranean fauna has been discovered. Genetic and morphological studies of subterranean dytiscid beetles and amphipods have suggested that individual calcretes are equivalent to closed island habitats, which have been isolated for millions of years. Here we test this ‘subterranean island’ hypothesis further by phylogeographic analyses of subterranean oniscidean isopods (Haloniscus), using mitochondrial DNA (mtDNA) sequence data derived from the cytochrome c oxidase subunit I gene. Phylogenetic and population genetic analyses provided evidence for significant phylogeographic structuring of isopod populations, with evidence for at least 24 divergent mtDNA lineages, each restricted in their distribution to a single calcrete aquifer. The high level of divergence among calcrete populations (generally >25%) and several mtDNA lineages within calcretes (>16%) suggests that each lineage is likely to represent a distinct species. These analyses, together with comparative phylogeographic data from dytiscid beetles and amphipods, provide strong support for the ‘subterranean island’ hypothesis, applying to both air-breathing and fully aquatic arthropod groups. The finding of several epigean lineages that grouped with stygobitic Haloniscus populations, and the overall phylogeographic structure of populations, suggests that the majority of stygobitic species evolved within individual calcretes following independent colonisation by epigean ancestors.