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To investigate the phylogeographic structure of the widespread freshwater prawn, Macrobrachium australiense, within and between major Australian drainage basins using mitochondrial sequence data. This will enable the investigation of historical connections between major drainages and examination of hypotheses of biogeographic associations among Australian freshwater basins. Location Inland, eastern and northern Australia. Methods Sequencing 16S rRNA and ATPase 6 protein coding mitochondrial DNA genes from M. australiense from 19 locations from inland, eastern and northern Australia. Results Within drainage basins, haplotype trees are monophyletic, with the exception of the Finke River from the Lake Eyre Basin. Macrobrachium australiense from the two main inland drainages, the Murray-Darling and Lake Eyre Basin are divergent from each other and do not form a monophyletic group, instead the Murray-Darling Basin haplotypes clade with eastern coastal haplotypes. Haplotypes from neighbouring eastern coastal drainages were found to be quite divergent from each other. Main conclusions The phylogeographic relationships among M. australiense suggest that the two major inland drainages, the Murray-Darling Basin and the Lake Eyre Basin, are not biogeographically closely associated to each other. Instead the Murray-Darling Basin is more closely allied with the eastern coastal drainages across the Great Dividing Range. Despite their proximity the neighbouring southeast Queensland coastal Mary and Brisbane Rivers are also biogeographically divergent from each other. The results also indicate that the Finke River appears to have been isolated from the remainder of the Lake Eyre Basin catchment for a significant period of time.