The genetic structure of the freshwater fish Ambassis macleayi Castelnau 1878 was explored across the tropical catchments of the Gulf of Carpentaria Basin, northern Australia. The Gulf of Carpentaria provides a unique opportunity to explore simultaneously contemporary and historical gene flow resulting from unique climatic patterns and historical connectivity among catchments via a freshwater lake that existed during lower sea levels. The control region of the mtDNA genome and 4 microsatellite loci were used to detect significant genetic structure among and within catchments. Within catchments, genetic structure suggested that dispersal of A. macleayi is restricted, despite high levels of connectivity during summer monsoonal events. Among catchments, divergence appeared to be deeper than what would be predicted based on the last opportunity for connectivity via the lake of Carpentaria (∼10,000 years before present [ybp]). Overall, these results have important implications for A. macleayi and other members of this genus. If individuals are not proficient dispersers, recolonization after disturbance will be limited. Because of historical isolation among catchments, each catchment harbors unique genetic diversity that should be conserved independently.