We examined genetic affinities of Aboriginal Australian and New Guinean populations by using nucleotide variation in the two hypervariable segments of the mtDNA control region (CR). A total of 318 individuals from highland Papua New Guinea (PNG), coastal PNG, and Aboriginal Australian populations were typed with a panel of 29 sequence-specific oligonucleotide (SSO) probes. The SSO-probe panel included five new probes that were used to type an additional 1,037 individuals from several Asian populations. The SSO-type data guided the selection of 78 individuals from Australia and east Indonesia for CR sequencing. A gene tree of these CR sequences, combined with published sequences from worldwide populations, contains two previously identified highland PNG clusters that do not include any Aboriginal Australians; the highland PNG clusters have coalescent time estimates of approximately 80,000 and 122,000 years ago, suggesting ancient isolation and genetic drift. SSO-type data indicate that 84% of the sample of PNG highlander mtDNA belong to these two clusters. In contrast, the Aboriginal Australian sequences are intermingled throughout the tree and cluster with sequences from multiple populations. Phylogenetic and multidimensional-scaling analyses of CR sequences and SSO types split PNG highland and Aboriginal Australian populations and link Aboriginal Australian populations with populations from the subcontinent of India. These mtDNA results do not support a close relationship between Aboriginal Australian and PNG populations but instead suggest multiple migrations in the peopling of Sahul.