As currently understood Monodelphis includes more than 22 species and is the most diverse genus of opossums (Didelphimorphia). No complete evaluation of the systematic relationships of its species has been attempted, despite the fact that several species groups and even genus-level groups have been proposed based on morphology, and that some of them are limited to recognized biogeographic regions. Here, genealogic relationships among 17 species were assessed based on phylogenetic analyses of 60 individual sequences (801 base pairs of the mitochondrial cytochrome b gene). The analyses cast doubts on the monophyly of Monodelphis, but species were consistently discriminated into eight species groups: (a) brevicaudata group, with five species, (b) adusta group, four species, (c) dimidiata group, two species, (d) theresa group, two species, (e) emiliae group, (f) kunsi group, (g) americana group, and (h) 'species C' group; the last four being monotypic. Genetic divergence among species within species groups ranges from 0.48 to 12.27%, and among species groups it goes from 15.61 to 20.15%. Further analyses in some species groups reveal congruence between genetic divergence, morphological traits, and geographic distribution, providing additional support for recognition of species limits. Although the cytochrome b gene may diverge too fast to evaluate relationships among the older lineages of the genus, the use of a broad taxon sampling allows for independent tests of hypotheses on species limits and relationships based on non-molecular characters. Congruent patterns offer a starting point for developing a sound taxonomy for Monodelphis and more robust hypotheses in regard to its diversification over many diverse Neotropical habitats.