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The influence of Pleistocene climatic cycles on Southern Hemisphere biotas is not yet well understood. Australia’s eastern coastal margin provides an ideal setting for examining the relative influence of landscape development, sea level fluctuation, and cyclic climatic aridity on the evolution of freshwater biodiversity. We examined the impact of climatic oscillations and physical biogeographic barriers on the evolutionary history of the wide-ranging Krefft’s river turtle (Emydura macquarii krefftii), using range-wide sampling (649 individuals representing 18 locations across 11 drainages) and analysis of mitochondrial sequences (similar to 1.3-kb control region and ND4) and nuclear microsatellites (12 polymorphic loci). A range of phylogeographic (haplotype networks, molecular dating), demographic (neutrality tests, mismatch distributions), and population genetic analyses (pairwise F-ST, analysis of molecular variance, Bayesian clustering analysis) were implemented to differentiate between competing demographic (local persistence vs. range expansion) and biogeographic (arid corridor vs. drainage divide) scenarios. Genetic data reveal population genetic structure in Krefft’s river turtles primarily reflects isolation across drainage divides. Striking north-south regional divergence (2.2% ND4 p-distance; c. 4.73Ma, 95% higher posterior density (HPD) 2.08-8.16Ma) was consistent with long-term isolation across a major drainage divide, not an adjacent arid corridor. Ancient divergence among regional lineages implies persistence of northern Krefft’s populations despite the recurrent phases of severe local aridity, but with very low contemporary genetic diversity. Stable demography and high levels of genetic diversity are inferred for southern populations, where aridity was less extreme. Range-wide genetic structure in Krefft’s river turtles reflects contemporary and historical drainage architecture, although regional differences in the extent of Plio-Pleistocene climatic aridity may be reflected in current levels of genetic diversity.