Phylogenetic relationships among 51 individuals of South American mouse opossums (Thylamys) were examined by using nucleotide sequence data from the mitochondrial cytochrome-b gene. Parsimony, likelihood, Bayesian, and genetic distance analyses revealed several distinct clades. T. macrurus (subtropical forest of eastern Paraguay) was strongly differentiated from all other species. T. venustus (Yungas forest on eastern slopes of the Andes of Bolivia and Argentina) separated into 2 strongly supported clades, providing support for recognition of 2 species, T. venustus and T. cinderella, but not supporting the distinctness of T. sponsorius. T. tatei (central coast of Peru) was sufficiently different from T. elegans (Matorral of Chile) to support species recognition. The dichotomy between northern (Chile and Bolivia) and southern (Argentina) clades of T. pallidior suggested the presence of 2 subspecies. High levels of DNA sequence variation indicated substantial genetic differentiation among and between clades, and combined with the phylogenetic analyses provide support for the systematic conclusions. Estimates of times since divergence suggest a radiation beginning in the Miocene and continuing through the Pliocene and correspond with changes in climate, vegetation, and geology that occurred during these times.