Koala (Phascolarctos cinereus) populations in southern Australia have a history of bottlenecks – earlier this century the species became extinct in South Australia, and almost so in Victoria. Subsequently large numbers of animals from island populations (founded from very few animals) have been translocated back to mainland sites and to other islands in the region. As part of a larger study of the genetic structure of koala populations in southern Australia, we have undertaken a survey of mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) variability. Genomic DNA from 91 koalas from five populations was examined using 23 restriction enzymes, and mtDNA fragments were detected using a domestic cat full-length mtDNA clone. Only one of the enzymes, TaqI, revealed polymorphism – a relatively low amount of variation compared with other mammals, although low mtDNA-RFLP variation has also been reported in Queensland koalas. French Island and populations established predominantly from French Island immigrant koalas, either directly or via other island populations, were indistinguishable by haplotype frequencies. The mtDNA data are thus consistent with the interpretation that the koala translocation programme has homogenized gene frequencies amongst those populations involved. South Gippsland is not recorded as having received translocated koalas directly, and has significantly different mtDNA-RFLP haplotype frequencies from all other populations examined. The fact that this distinction was not previously observed in nuclear gene frequencies may reflect predominantly male-mediated dispersal in koalas.