The gray mouse opossum (Tlacuatzin canescens) is a poorly studied marsupial endemic to México. Phylogenetic or phylogeographic information on this species and its evolutionary history is scarce and scattered. We investigated the origin and the diversification of this taxon using molecular and morphological data. We examined mitochondrial (cytochrome-b) and nuclear (interphotoreceptor retinoid-binding protein) sequences to test species monophyly and assess phylogenetic relationships within the species. We also used multivariate statistical analyses of 13 craniodental variables to assess the phenotypic distinctiveness of mitochondrial haplotypic clades. Phylogenetic analyses revealed that T. canescens is a monophyletic group divided into 5 well-differentiated clades. Genetic divergence among clades was 3.1–8.9%, while the intragroup divergence was 0.5–3.7%. Our molecular clock estimates indicate that the diversification of this taxon occurred during the Pleistocene, which suggests that climatic changes and the presence of geographical barriers influenced the genetic differentiation of its populations. Morphological analyses also showed that there are cranial differences among these clades. Our results suggest the recognition of T. gaumeri, T. insularis, and T. sinaloae as valid species, and the recognition of 1 clade that we describe here as a new species.