This study used allozymes and mtDNA variation to test that: 1) populations of two fish species, Nematolosa erebi and Retropinna semoni, in lowland rivers in central Australia were highly connected within drainages, 2) populations in different drainages were highly differentiated and 3) there was evidence of historical connections between two major lowland drainages in inland Australia. Levels of genetic differentiation among populations within drainages were low, but still statistically significant, indicating that populations were not as highly connected as had been predicted. Populations from the Murray–Darling and the Lake Eyre drainages were highly differentiated, indicating no contemporary dispersal across drainage boundaries. Both species showed evidence of historical connections between the two drainage basins, although estimates of the time that these last occurred differed between the two species. Nematolosa erebi populations from the two drainages were estimated to have been separated c. 150 000 years ago, whereas populations of R. semoni, were estimated to have been separated c. 1.5 million years ago.