Conservation programs for threatened species are greatly benefiting from genetic data, for their power in providing knowledge of dispersal/gene flow across fragmented landscapes and for identifying populations of high conservation value. The endangered southern brown bandicoot (Isoodon obesulus obesulus) has a disjunct distribution range in South Australia, raising the possibility that populations of the subspecies may represent distinct conservation units. In the current study, we used a combination of 14 microsatellite and two mitochondrial sequence markers to investigate the phylogeography and population structure of I. o. obesulus in South Australia and south-western Victoria, with the aim of identifying any potential evolutionarily significant units and management units relevant to conservation management. Our phylogenetic/population analyses supported the presence of two distinct evolutionary lineages of I. o. obesulus. The first lineage comprised individuals from the Mount Lofty Ranges, Fleurieu Peninsula and Kangaroo Island. A second lineage comprised individuals from the south-east of South Australia and south-western Victoria. We propose that these two lineages represent distinct evolutionarily significant units and should be managed separately for conservation purposes. The findings also raise significant issues for the national conservation status of I. o. obesulus and suggest that the current subspecies classification needs further investigation.