The largest genus of salamanders, Bolitoglossa (Plethodontidae), is widespread in tropical America, where it occurs in diverse habitats and elevations, from high elevation grasslands to lowland rain forest. It has the most extensive geographical range of any salamander genus. While most species occur in Middle America, it ranges throughout most of tropical South America as well. Phylogenetic analysis of 1196 bp of two mitochondrial genes (cytochrome b, 16S RNA) from 55 species offers strong support for the monophyly of the genus and sorts the species into a number of clades. Taking into account morphology, distribution, general ecology, and prior systematic and taxonomic studies, we recognize seven subgenera, four of them new: Bolitoglossa Dumeril, Bibron et Dumeril, 1854, Eladinea Miranda Ribeiro, 1937, Magnadigita Taylor, 1944, Mayamandra, Nanotriton, Oaxakia and Pachymandra. All South American and some lower Middle American species are included in a single well supported clade, Eladinea. At the species level our analyses uncover the existence of large genetic diversity within morphologically homogeneous taxa. We propose the new combination: B. (Eladinea) paraensis (Unterstein, 1930) stat. nov., for Brazilian salamanders previously included under B. altamazonica. We evaluate evidence for the multiple colonization of the tropical lowlands by morphologically derived species groups. South America was invaded by members of one clade, Eladinea, which we infer to have dispersed to South America prior to closure of the Panamanian Portal. Despite the relatively long history of salamanders in South America, that continent now accounts for a relatively small proportion of the lineages and species of neotropical salamanders.