Aim: We conducted a range-wide phylogeographic study of a common Australian freshwater fish, the golden perch (Macquaria ambigua), to investigate the relationship between environmental processes and evolutionary history in drainage basins. Location: Inland [Lake Eyre (LEB), Murray–Darling (MDB) and Bulloo (BULL)] and coastal basins [Fitzroy (FITZ)] of eastern Australia. Methods: A total of 590 samples were collected from across the entire species’ distribution and a section of the mitochondrial DNA control region was sequenced. In order to reconstruct the evolutionary history of M. ambigua a comprehensive suite of phylogeographic analyses was conducted, including nested clade phylogeographic analysis, mismatch analysis and isolation-with-migration model simulations. Results: Three major lineages corresponding to the major drainage basins, FITZ, MDB and LEB/BULL, were identified (ΦST = 0.92). Lineages from the coastal basin (FITZ) were highly divergent from those of the inland basins (up to 6%). Levels of genetic diversity in the inland basins were relatively low and our analyses indicate that these populations experienced both demographic and range expansions during the Pleistocene. Main conclusions: Investigation of the range-wide phylogeography of M. ambigua has revealed new insights into the biogeography of the Australian arid zone, particularly with regard to evolutionary events chronologically associated with cyclical moist and dry conditions. We propose that M. ambigua originated on the east coast (FITZ) and crossed a major geographic barrier, the Great Dividing Range (GDR), to colonize the inland basins (MDB, LEB and BULL). We infer a series of demographic and range expansion events for M. ambigua consistent with a scenario of moister Pleistocene conditions and increased connectivity of freshwater environments, both within and among drainage basins. Major lineages then diversified following isolation of freshwater environments under increasingly arid climate conditions. We suggest that management priorities for M. ambigua should include the resolution of taxonomic uncertainties and the maintenance of genetic diversity of both stocked populations in the MDB and native populations of the LEB that may be at risk of further isolation and reduced gene flow due to increased aridification under future climate change scenarios.