Relationships of 5 families within traditional Yangochiroptera (Furipteridae, Mystacinidae, Myzopodidae, Natalidae, and Thyropteridae) have been debated considerably for more than a century, resulting in several alternative hypotheses for their evolution and zoogeography. A recent cladistic assessment of diverse morphologic traits and another of mitochondrial deoxyribonucleic acid (mtDNA) sequences have contradicted both traditional classification of yangochiropterans and each other. Our goals were to test alternative phylogenetic hypotheses by examining DNA sequences (1.4 kb) from a nuclear gene (recombination-activating gene-2; Rag2) and, if appropriate, to examine family-level relationships based on concatenation of Rag2 and mtDNA sequences (4.1 kb). Relationships suggested by parsimony and Bayesian analysis of Rag2 data were congruent with mtDNA, and combined analysis afforded high statistical support for all but 2 clades. These molecular data strongly support Noctilionoidea (Furipteridae, Mormoopidae, Mystacinidae, Noctilionidae, Phyllostomidae, and Thyropteridae) and Vespertilionoidea (Molossidae, Natalidae, and Vespertilionidae) and strongly contradict traditional association between Myzopodidae and other noctilionoid or vespertilionoid families. This study, current distributions, and limited fossil record strengthen the hypothesis of a New World origin for Noctilionoidea and suggest an Old World origin for Myzopodidae. Despite considerable statistical support afforded by these molecular data, further study using extensive taxonomic sampling of all bat families is needed to fully resolve relationships among Myzopodidae, Noctilionoidea, and Vespertilionoidea.