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Knowledge of genetic structure and patterns of connectivity is valuable for implementation of effective conservation management. The arid zone of Australia contains a rich biodiversity, however this has come under threat due to activities such as altered fire regimes, grazing and the introduction of feral herbivores and predators. Suitable habitats for many species can be separated by vast distances, and despite an apparent lack of current geographical barriers to dispersal, habitat specialisation, which is exhibited by many desert species, may limit connectivity throughout this expansive region. We characterised the genetic structure and differentiation of the great desert skink (Liopholis kintorei), which has a patchy, but widespread distribution in the western region of the Australian arid zone. As a species of cultural importance to local Aboriginal groups and nationally listed as Vulnerable, it is a conservation priority for numerous land managers in central Australia. Analysis of mitochondrial ND4 sequence data and ten nuclear microsatellite loci across six sampling localities through the distribution of L. kintorei revealed considerable differentiation among sites, with mitochondrial FST and microsatellite F′ST ranging from 0.047-0.938 and 0.257-0.440, respectively. The extent of differentiation suggests three main regions that should be managed separately, in particular the southeastern locality of Uluru. Current genetic delineation of these regions should be maintained if future intervention such as translocation or captive breeding is to be undertaken.