Pennyfish, Denariusa bandata, are small freshwater fish widely distributed in northern Australia in 4 highly disjunct regions and in the Fly River and Bensbach River drainages in southern Papua New Guinea (PNG). Alternating phases of exposure of the Australia–New Guinea Continental Shelf (ANGCS) during stands of lowered sea levels during Pleistocene glacial phases created a land bridge and fresh/brackish water habitats that intermittently connected Australia and PNG. Some biogeographic theories and empirical evidence for several freshwater crustaceans suggest that wetlands on the exposed ANGCS during the last glacial maximum were an important conduit for dispersal and gene flow in freshwater species between Australia and PNG and across northern Australia and that connectivity was severed by the most recent sea level rise in the region (∼6–8 thousand years before present). We used mitochondrial deoxyribonucleic acid (mtDNA) sequence data to test phylogeographic hypotheses concerning the origin of the disjunct distribution in D. bandata in relation to dispersal–vicariant processes and exposure of the ANGCS during Pleistocene sea level changes. Rather than a late Pleistocene origin associated with the last glacial maximum, the coalescence of the regional populations was in the early- to mid-Pleistocene, and the molecular data indicated that the eastern regional populations split before the northern populations split (i.e., sequential vicariance), whereas the northern populations split contemporaneously (i.e., simultaneous vicariance). The complex mtDNA genealogy for D. bandata also indicated a phylogeographic history in which ancestral lineages were retained in the northwestern part of its distribution, and ancestral haplotype diversity was retained in the Fly River (PNG) population because several divergent clusters of PNG haplotypes were more closely related to Australian haplotypes than to each other.