Phylogeography is a field that has the potential to provide an integrative approach to the conservation of threatened species. The southern purple spotted gudgeon, Mogurnda adspersa, is a small freshwater fish that was once common and widely distributed throughout south-eastern Australia. However, habitat alteration has dramatically reduced the size and the range of Murray–Darling Basin populations, which are now classified as endangered. Here patterns of genetic structure and evolutionary history of M. adspersa in southern Queensland and the Murray–Darling Basin are elucidated using three regions of the mitochondrial DNA, the ATPase 6 and 8 and the control region. Murray–Darling Basin populations are characterised by lineages with highly localised endemism, very low genetic diversity and restricted gene flow. Phylogenetic reconstructions show that Murray–Darling Basin populations comprise a monophyletic clade that possibly originated by range expansion from the coast around 1.6 million years ago. It is proposed that the divergent Murray–Darling Basin clade is of high conservation priority and requires separate management. The present study further exemplifies the role of drainage rearrangement in driving evolutionary diversification in Australian freshwater fishes, an historical process with profound implications for conservation management.