Monodelphis (short-tailed opossums) is the most speciose South American marsupial genus, wtih species distributed from Panama to Argentina. The goal of the present study was to analyze levels and patterns of genetic variation in M. brevicaudata (Guianan short-tailed opossum), M. dimidiata (yellow-sided opossum), M. domestica (gray short-tailed opossum), and M. kunsi (pygmy short-tailed opossum) using sequences of the mitochondrial cytochrome b (Cytb) and nuclear interphotoreceptor retinoid-binding protein (IRBP) genes from individuals sampled in the Amazon, Cerrado, Caatinga, Atlantic Forest, and Pampa biomes in Brazil. Species relationships also were analyzed using sequences of 9 other Monodelphis species retrieved from GenBank and representing 3 localities in Brazil and 17 sites in other South American countries. M. domestica and M. brevicaudata showed phylogeographic structure, but M. dimidiata and M. kunsi did not. Maximum parsimony, maximum likelihood, and Bayesian algorithms showed similar topologies for estimated phylogenetic trees. Analyses of individual genes produced comparable results, but combined loci produced trees with higher support. All Monodelphis haplotypes were monophyletic, with M. emiliae (Emilia's short-tailed opossum) sister to all other species, which form 2 groups. M. brevicaudata (from the Amazon) forms a well-supported clade with M. domestica (which occupies several biomes) and 2 unidentified Amazonian species. The other branch has M. kunsi (from the Cerrado) as sister to a well-supported clade of 5 Amazonian species.