The biogeography of southern Australia is characterized by a repeated pattern of relatedness between the biota of southwestern and southeastern Australia. Both areas possess a temperate climate but are separated by a vast arid region, currently lacking permanent freshwater habitats, which has become increasingly drier since about 15 Ma. Aquatic organisms have thus potentially remained isolated for a considerable time. Pygmy perches (Nannatherina and Nannoperca, Percichthyidae) provide an excellent scenario for investigating biogeographic relationships between southwestern and southeastern regions as multiple species occur on either side of Australia. This allows us to potentially differentiate between “Multiple Invasion” and “Endemic Speciation,” the two major hypotheses proposed to account for current distributions. The first suggests that multiple east–west movements have occurred, whereas the second suggests a single east–west split, with current biodiversity in each region being reciprocally monophyletic. Systematic relationships within this group were investigated with the mitochondrial cytochrome b gene; nuclear intron and exon sequences from S7, RAG1, and RAG2; and 53 allozyme loci. Our data supported the hypothesis of multiple movements across southern Australia based on a consistent lack of support for reciprocal monophyly of eastern and western species. This study appears to be the first example of an animal group displaying clear multiple east–west movement in southern Australia, as all other aquatic and terrestrial fauna previously examined displayed a single east–west split. Despite a high degree of sympatry within each region, the only evidence for hybridization was found between Nannoperca australis and N. obscura, with the latter having its mitochondrial genome completely replaced by that of N. australis, with no evidence for nuclear introgression. This is one of only a few confirmed examples of complete replacement of the mitochondrial genome in one species with that of another. Cryptic differentiation was also evident within the two most widespread species, N. australis and N. vittata, indicating that these likely consist of multiple species. We also highlight the need for multiple molecular markers with different strengths in order to obtain a more robust phylogeny, despite problems resulting from potential incongruences between data sets.