A detailed phylogeny of bumblebees is urgently needed to understand speciation and biogeographic diversification in the Neotropical region. We sequenced autosomal and mtDNA loci from nine Brazilian bumblebee species and compiled it with the data already available to obtain highly resolved phylogenetic trees with fossil-calibrated dates. The ancestral Bombus lineage was estimated to diversify between 47.08 and 34.27 million years ago (Ma) in the Holarctic region, but largely restricted to the eastern Old World. The Neotropical region was initially colonized in the Late Miocene, where bumblebee diversification was shown to be consistent with geologic and climatic events of the Late Cenozoic. Neotropical bumblebees likely originated from Nearctic lineages, which dispersed towards South America after 29 Ma.