Use of DNA from museum samples is a powerful tool to directly establish historical ESU boundaries in areas where populations of a species have been extirpated. The brush-tailed rock-wallaby (Petrogale penicillata) has suffered extensive reductions in range and population size since European settlement in Australia. Populations of this species have been grouped into three putative evolutionarily significant units (ESUs) which were likely once contiguous along the south-east coast of Australia. However, there is currently a gap of ~320 km between extant populations of the southern and central ESUs. Conservation plans for the brush-tailed rock-wallaby include re-introductions of animals to locations within their former range. In order to preserve the historic geographic integrity of the genetic lineages within the species, it is necessary to map the boundary between the southern ESU and central ESU to allow more informed management decisions about which lineages should be used to re-stock specific geographic locations. We have extracted DNA from samples from museum specimens that come from locations between the southernmost extant colony of the central ESU and the remaining wild colony of the southern ESU. We sequenced a 177 bp interval of the left domain of the mitochondrial DNA control region and used phylogenetic analysis to group obtained sequences with previously published sequences belonging to the three ESUs. We have extended the range of the central ESU southwards and the range of the southern ESU northwards such that the gap between the ESUs is now approximately 160 km. We recommend that, where suitable historical museum collections exist, this technique be incorporated into re-introduction plans for other threatened species.