The complete mitochondrial (mt) genomes of five marsupial species have been sequenced. The species represent all three South American orders (Didelphimorphia, Paucituberculata, and Microbiotheria). Phylogenetic analysis of this data set indicates that Didelphimorphia is a basal marsupial lineage followed by Paucituberculata. The South American microbiotherid Dromiciops gliroides (monito del monte) groups with Australian marsupials, suggesting a marsupial colonization of Australia on two occasions or, alternatively, a migration of an Australian marsupial lineage to South America. Molecular estimates suggest that the deepest marsupial divergences took place 64-62 million years before present (MYBP), implying that the radiation of recent marsupials took place after the K/T (Cretaceous/Tertiary) boundary. The South American marsupial lineages are all characterized by a putatively non-functional tRNA for lysine, a potential RNA editing of the tRNA for asparagine, and a rearrangement of tRNA genes at the origin of light strand replication.