The characterization of the different taxa of the highly diverse genus Monodelphis is poorly understood, as is the case of their distribution. Historically, taxonomic studies of Monodelphis have been restricted to a few or single species, whereas molecular approaches have been used for estimating phylogenetic relationships between species. We carried out phylogenetic analyses of 14 Monodelphis species, including Monodelphis domestica, based on cytochrome b mitochondrial DNA sequences. Forty-five complete (1149 bp) sequences of this gene were obtained from 39 specimens of M. domestica collected in 23 localities of the Brazilian Cerrado, Pantanal, and Caatinga morphoclimatic domains; one of Monodelphis umbristriata, two of Monodelphis americana, and two of Monodelphis dimidiata. A total of 72 haplotypes were analyzed, 48 only in M. domestica. Analyses were carried out in conjunction with 46 other sequences retrieved from GenBank, including M. domestica, Monodelphis brevicaudata, Monodelphis glirina, Monodelphis emiliae, Monodelphis peruviana, Monodelphis osgoodi, Monodelphis handleyi, Monodelphis kunsi, Monodelphis americana, Monodelphis dimidiata, Monodelphis iheringi, Monodelphis reigi, and Monodelphis adusta, with six other different didelphid species used as outgroups. Maximum likelihood and Bayesian inference were similar in depicting phylogenetic relationships of different Monodelphis taxa. Two clades of M. domestica were defined on the basis of these results. Genetic distance estimates ranged from 3.2% to 6.2% between these clades of M. domestica. Population analyses were carried out to infer the likely demographic scenarios and the relationship between M. domestica haplotypes. Median-joining and spatial analyses showed two populations related to different morphoclimatic domains (Cerrado/Pantanal and Caatinga). These results indicate a population structure in M. domestica and the possibility that this taxon might represent a species complex.