The genus Thylamys Gray, 1843 lives in the central and southern portions of South America inhabiting open and shrub-like vegetation, from prairies to dry forest habitats in contrast to the preference of other Didelphidae genera for more mesic environments. Thylamys is a speciose genus including T. elegans (Waterhouse, 1839), T. macrurus (Olfers, 1818), T. pallidior (Thomas, 1902), T. pusillus (Desmarest, 1804), T. venustus (Thomas, 1902), T. sponsorius (Thomas, 1921), T. cinderella (Thomas, 1902), T. tatei (Handley, 1957), T. karimii (Petter, 1968), and T. velutinus (Wagner, 1842) species. Previous phylogenetic analyses in this genus did not include the Brazilian species T. karimii, which is widely distributed in this country. In this study, phylogenetic analyses were performed to establish the relationships among the Brazilian T. karimii and all other previously analyzed species. We used 402-bp fragments of the mitochondrial cytochrome b gene, and the phylogeny estimates were conducted employing maximum parsimony (MP), maximum likelihood (ML), Bayesian (BY), and neighbor-joining (NJ). The topologies of the trees obtained in the different analyses were all similar and pointed out that T. karimii is the sister taxon of a group constituted of taxa from dry and arid environments named the dryland species. The dryland species consists of T. pusillus, T. pallidior, T. tatei, and T. elegans. The results of this work suggest five species groups in Thylamys. In one of them, T. velutinus and T. kariimi could constitute a sister group forming one Thylamys clade that colonized Brazil.