Southern brown (Isoodon obesulus) and golden (Isoodon auratus) bandicoots are iconic Australian marsupials that have experienced dramatic declines since European settlement. Conservation management programs seek to protect the remaining populations; however, these programs are impeded by major taxonomic uncertainties. We investigated the history of population connectivity to inform subspecies and species boundaries through a broad-scale phylogeographic and population genetic analysis of Isoodon taxa. Our analyses reveal a major east–west phylogeographic split within I. obesulus/I. auratus, supported by both mtDNA and nuclear gene analyses, which is not coincident with the current species or subspecies taxonomy. In the eastern lineage, all Tasmanian samples formed a distinct monophyletic haplotype group to the exclusion of all mainland samples, indicative of long-term isolation of this population from mainland Australia and providing support for retention of the subspecific status of the Tasmanian population (I. o. affinis). Analyses further suggest that I. o. obesulus is limited to south-eastern mainland Australia, representing a significant reduction in known range. However, the analyses provide no clear consensus on the taxonomic status of bandicoot populations within the western lineage, with further analyses required, ideally incorporating data from historical museum specimens to fill distributional gaps.