Gesneriaceae are represented in the New World (NW) by a major clade (c. 1000 species) currently recognized as subfamily Gesnerioideae. Radiation of this group occurred in all biomes of tropical America and was accompanied by extensive phenotypic and ecological diversification. Here we performed phylogenetic analyses using DNA sequences from three plastid loci to reconstruct the evolutionary history of Gesnerioideae and to investigate its relationship with other lineages of Gesneriaceae and Lamiales. Our molecular data confirm the inclusion of the South Pacific Coronanthereae and the Old World (OW) monotypic genus Titanotrichum in Gesnerioideae and the sister-group relationship of this subfamily to the rest of the OW Gesneriaceae. Calceolariaceae and the NW genera Peltanthera and Sanango appeared successively sister to Gesneriaceae, whereas Cubitanthus, which has been previously assigned to Gesneriaceae, is shown to be related to Linderniaceae. Based on molecular dating and biogeographical reconstruction analyses, we suggest that ancestors of Gesneriaceae originated in South America during the Late Cretaceous. Distribution of Gesneriaceae in the Palaeotropics and Australasia was inferred as resulting from two independent long-distance dispersals during the Eocene and Oligocene, respectively. In a short time span starting at 34 Mya, ancestors of Gesnerioideae colonized several Neotropical regions including the tropical Andes, Brazilian Atlantic forest, cerrado, Central America and the West Indies. Subsequent diversification within these areas occurred largely in situ and was particularly extensive in the mountainous systems of the Andes, Central America and the Brazilian Atlantic forest. Only two radiations account for 90% of the diversity of Gesneriaceae in the Brazilian Atlantic forest, whereas half of the species richness in the northern Andes and Central America originated during the last 10 Myr from a single radiation.