A phylogenetic analysis of the intrageneric relationships within the bee genus Colletes using a combined data set of the mitochondrial cytochrome oxidase 1 (COI) and 28S sequences is presented and its implications for historical biogeography are investigated. We analyzed 91 out of 469 described Colletes species (19.4%) from the entire range of distribution and 25 outgroup taxa representing all colletid subfamilies closely related to Colletinae. Eight different combinations of alignment parameters were used for the 28S data, and our combined data set comprised between 1801 and 1845 aligned nucleotides with COI contributing 339 informative bases and 28S between 318 and 360 informative sites. Our results corroborate the monophyly of about half of the Old World subgenera and show the need for a taxonomic re-delimitation of other groups currently accepted. The phylogenetic results confirm the South American origin of Colletes and multiple faunal exchange events between North America and Eurasia. The last continent colonized was Africa south of the Sahara, which experienced at least two independent invasions.