Several freshwater fishes have disjunct (geographically discrete and widely spaced) distributions across northern Australia. We used mitochondrial deoxyribonucleic acid (mtDNA) data and phylogeographic analyses to examine the origin of these common disjunct distributions and to test the hypothesis that they were a result of a single biogeographic event. These disjunct distributions are not perfectly shared among species, but we selected 3 species that have wide ranges with the most similar disjunct geographic overlap: Spotted Blue Eye (Pseudomugil gertrudae), Pennyfish (Denariusa australis), and MacCulloch's Rainbow Fish (Melanotaenia maccullochi). Despite similarity in their present-day disjunct distributions, spatial genetic patterns varied considerably among the 3 species in terms of measures of molecular diversity, number of mtDNA lineages within each species, inter-and intra-regional spatial distribution of individual lineages within species, and degree of partitioning of genetic variation among regions. Pseudomugil gertrudae and D. australis each contained 1 to 2 divergent lineages at particular sites in 1 of the regions (Top End), but both species also contained lineages in this region that were more closely related to conspecific populations in other regions. Two regional populations (Top End and Northern Cape York Peninsula) of M. maccullochi consisted exclusively of highly divergent lineages that probably reflect cryptic species. When the divergent lineages within each species were excluded from temporal analyses, a single vicariant event among regions could not be rejected. Our results indicate that several regional populations are long-term relicts for M. maccullochi and that several sites within Top End are associated with localized long-term refugia for P. gertrudae and D. australis. A single biogeographic event in the mid-to late-Pleistocene may have created broadscale separation of most populations of these species.