Building on the recent publication of the first monotreme genome, that of the platypus, and the discovery that many platypus microsatellites are found in the genomes of three mammals (opossum, human, mouse) and two non-mammalian vertebrates (chicken, lizard), we investigated further the evolutionary conservation of microsatellites identified in the monotreme lineage and tested whether the conservation of microsatellites we observe in vertebrates has phylogenetic signal. Most conserved platypus microsatellites (75%) were found in one species, with the platypus sharing many more microsatellites with mammals than with reptiles (83% versus 30%). Within mammals, unexpectedly, many more platypus microsatellites had orthologues in the opossum genome than in that of either human or mouse, which was at odds with the very well supported view that monotremes diverged from a lineage containing both eutherians and marsupials (Theria hypothesis). We investigated the phylogenetic significance of microsatellite conservation through Bayesian and maximum parsimony tree reconstruction using presence/absence data of microsatellite loci conserved in a total of 18 species, including the platypus. Although models of evolution implemented in current phylogenetic reconstruction algorithms are not tailor-made for microsatellite data, we were able to construct vertebrate phylogenies that correspond well to the accepted mammalian phylogeny, with two of our three reconstructions supporting the Theria hypothesis. Our analysis provides ground for new theoretical development in phylogeny-based analyses of conserved microsatellite data.