DNA sequences from the mitochondrial cytochrome b gene were obtained from a museum specimen of the presumed extinct thylacine (Thylacinus cynocephalus) and were compared with homologous sequences from 13 representatives of the Australian marsupial family Dasyuridae. The relationship of the thylacine to dasyurids has been suggested by previous anatomical and molecular studies, but its position within the dasyuroid radiation has not been addressed with genetic data. Phylogenetic analysis of the sequences reported here suggests that the thylacine is a sister group to Dasyuridae and lends support to the hypothesis that Thylacinus represents an ancient Australian marsupial lineage. Relationships with Dasyuridae support the results of other recent molecular studies, particularly in showing the affinities of endemic New Guinean subfamilies with larger Australian clades.