1. Flow regime and riverine architecture are two important landscape characteristics that influence genetic diversity and gene flow in riverine species. 2. Using population genetic markers (mtDNA, microsatellites and allozymes), this study aimed to investigate genetic diversity and gene flow in the freshwater fish, Neosilurus hyrtlii, across two major drainage divisions in northern and central Australia (the Gulf of Carpentaria and Lake Eyre basins). These basins lie adjacent to each other and differ in their hydrological inputs and riverine structure, providing an ideal opportunity to identify the impact of landscape processes upon population dynamics of freshwater fish. 3. Populations were strongly structured among basins, among catchments within basins and were weakly structured within catchments in the Lake Eyre Basin, providing support for the Stream Hierarchy Model. 4. Interestingly, mtDNA and microsatellite diversity was much higher in the Gulf of Carpentaria Basin compared to the Lake Eyre Basin. It was concluded that this difference was due to the extreme hydrological variability in this basin and boom-bust population