The complete mitochondrial DNA (mtDNA) (16,896 nt) of the wallaroo (Macropus robustus) was sequenced. The concatenated amino acid sequences of 12 mitochondrial protein-coding genes of the wallaroo plus those of a number of other mammals were included in a phylogenetic study of early mammalian divergences. The analysis joined monotremes and marsupials (the Marsupionta hypothesis) to the exclusion of eutherians. The analysis rejected significantly the commonly acknowledged Theria hypothesis, according to which Marsupialia and Eutheria are grouped together to the exclusion of Monotremata. The region harboring the gene for lysine tRNA (tRNA-Lys) in the mtDNA of other vertebrates is in the wallaroo occupied by a sequence (tRNA-Lys) that lacks both an anticodon loop as well as the anticodon for the amino acid lysine. An alternative tRNA-Lys gene was not identified in any other region of the mtDNA of the wallaroo, suggesting that a tRNA-Lys of nuclear origin is imported into marsupial mitochondria. Previously described RNA editing of tRNA-Asp and rearrangement of some tRNA genes were reconfirmed in the mtDNA of the wallaroo. The divergence between Monotremata/Marsupialia and Eutheria was timed to approximately 130 million years before present (MYBP). The same calculations suggested that Monotremata and Marsupialia diverged approximately 115 MYBP and that Australian and American marsupials separated approximately 75 MYBP. The findings also show that many, probably most, extant eutherian orders had their origin in middle to late Cretaceous times, 115-65 MYBP.