We examined the influence of past and present landscape structure across five regions of northern and eastern Australia on phylogeographic structure in the freshwater fish Glossamia aprion. We predicted the geological isolation of North West Australia would promote the strongest phylogeographic structure, including highest endemism of candidate species and deepest divergences within species, and past low sea level riverine connections in the Carpentaria Region would greatly reduce phylogeographic structure. The allozyme data indicated five candidate species within Australian Glossamia, although the mtDNA data revealed only four of these taxa. Diversity and endemism of the candidate species was highest in North West Australia, but Carpentaria was not the only region to have low species diversity or an absence of endemic species. Three of the five candidate species were narrow range endemics and occurred in regions with known endemism of candidate species in other freshwater fauna. The reported subspecies Glossamia aprion gillii was clearly distinct based on the genetic data, while the fifth species, G. a. aprion, was widely distributed across four of the five regions. The intra-specific analyses for G. a. aprion showed highest phylogeographic structure in North West Australia and lowest phylogeographic structure in Carpentaria. Overall, the results demonstrate the long-term legacy of variable landscape structure on phylogeographic patterns and allopatric speciation.