The spotted galaxias (or trout minnow), Galaxias truttaceus, is a species that is restricted to south-western and south-eastern Australia, but there has long been conjecture as to whether the geographically and Critically Endangered Western Australian populations represent a subspecies (Galaxias truttaceus hesperius). We provide evidence that Western Australian populations, on the basis of a combination of genetic, geographic and ecological criteria, should be considered an evolutionary significant unit, which merits management as a high conservation priority. Substructure at nuclear and matrilineal genetic markers is not suggestive of species-level divergence, but rather of discrete western and eastern Australian subpopulations with limited contemporary gene flow. In contrast to many eastern populations that are diadromous, all western populations are potamodromous. Adults live and spawn in riverine habitats and larvae drift downstream to coastal lakes, where they spend several months, before undertaking a distinct upstream recruitment migration as juveniles to colonise riverine habitats. Instream barriers that disconnect riverine and lentic habitats restrict distributional range and presumably affect reproductive success of Western Australian populations. Conserving the remaining populations in Western Australia will require ongoing efforts to reduce the impact of emerging threats, particularly those related to instream barriers, introduced species and reductions in water quantity and quality.