Despite only limited Pleistocene glacial activity in the southern hemisphere, temperate forest species experienced complex distributional changes resulting from the combined effects of glaciation, sea level change and increased aridity. The effects of these historical processes on population genetic structure are now overlain by the effects of contemporary habitat modification. In this study, 10 microsatellites and 629 bp of the mitochondrial control region were used to assess the effects of historical forest fragmentation and recent anthropogenic habitat change on the broad-scale population genetic structuring of a southern temperate marsupial, the Tasmanian pademelon. A total of 200 individuals were sampled from seven sites across Tasmania and two islands in Bass Strait. High mitochondrial and nuclear genetic diversity indicated the maintenance of large historical population sizes. There was weak phylogeographical structuring of haplotypes, although all King Island haplotypes and three Tasmanian haplotypes formed a divergent clade implying the mid-Pleistocene isolation of a far northwestern population. Both the mitochondrial and nuclear data indicated a division of Tasmanian populations into eastern and western regions. This was consistent with a historical barrier resulting from increased aridity in the lowland ‘midlands’ region during glacial periods, and with a contemporary barrier resulting from recent habitat modification in that region. In Tasmania, gene flow appears to have been relatively unrestricted during glacial maxima in the west, while in the east there was evidence for historical expansion from at least one large glacial refuge and recolonization of Flinders Island.