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Musk Ducks (Biziura lobata) are endemic to Australia and occur as two geographically isolated populations separated by the Nullarbor Plain, a vast arid region in southern Australia. We studied genetic variation in Musk Duck populations at coarse (eastern versus western Australia) and fine scales (four sites within eastern Australia). We found significant genetic structure between eastern and western Australia in the mtDNA control region (I broken vertical bar(ST) = 0.747), one nuclear intron (I broken vertical bar(ST) = 0.193) and eight microsatellite loci (F(ST) = 0.035). In contrast, there was little genetic structure between Kangaroo Island and adjacent mainland regions within eastern Australia. One small population of Musk Ducks in Victoria (Lake Wendouree) differed from both Kangaroo Island and the remainder of mainland eastern Australia, possibly due to genetic drift exacerbated by inbreeding and small population size. The observed low pairwise distance between the eastern and western mtDNA lineages (0.36%) suggests that they diverged near the end of the Pleistocene, a period characterised by frequent shifts between wet and arid conditions in central Australia. Our genetic results corroborate the display call divergence and Mathews’ (Austral Avian Record 2:83-107, 1914) subspecies classification, and confirm that eastern and western populations of Musk Duck are currently isolated from each other.