The Australian Monsoonal Tropics (AMT) is a unique location for the study of phylogeography and intraspecific genetic variation in freshwater fish. We assessed the phylogeographic structure of 5 species from 2 genera across the region. The species included 3 neosilurids (Plotosidae, Neosilurus hyrtlii, Neosilurus ater, and Neosilurus pseudospinosus) and 2 members of the genus Oxyeleotris (Eleotridae, O. selheimi and O. lineolata). We used mitochondrial deoxyribonucleic acid (mtDNA) and phylogenetic analyses to explore the phylogeographic histories of these species. Overall, phylogeographic patterns were inconsistent. Some species were highly structured, and phylogeographic breaks were detected (e.g., N. hyrtlii, N. pseudospinosus, and O. selheimi), but other species showed no obvious divergences across the AMT (N. ater and O. lineolata). All species sampled in the Gulf of Carpentaria had shallow phylogenies, consistent with the expectation that historically, Lake Carpentaria would have provided connectivity through this region. All species also showed evidence of recent connectivity across drainage divides on the eastern and western coasts of the Cape York Peninsula. Some species in the Kimberley region were highly structured, consistent with expectation that these ancient and geologically stable catchments would promote divergence in allopatry. Conservation efforts should now be directed toward ensuring that the intraspecific biodiversity identified in our study and others are protected in the future.