We estimated phylogenetic relationships among species of the bee genus Diadasia, a group of new world, specialist bees. We sequenced approximately 2 kb of the mitochondrial genes cytochrome oxidase subunit I and II and tRNA leucine and approximately 1 kb of the nuclear gene elongation factor 1-alpha for 24 North American Diadasia species, 4 South American species, and five outgroup genera. Parsimony analyses of the two data sets were highly congruent. A combined analysis produced a well-resolved phylogenetic hypothesis that supported the monophyly of Diadasia, but not that of traditional subgenera: Diadasia s. str. was paraphyletic in all analyses. With one exception, the North and South American species formed separate clades, supporting previous hypotheses that two lineages of Diadasia have dispersed from South to North America: a more recent dispersal of D. ochracea and an older dispersal of the ancestor to all other North American species. Different species of Diadasia specialize on pollen from at least five different plant families; the phylogeny presented here, along with known host affinities, indicates that host-switching has been rare.