Molecular phylogenetic analyses based on expanded taxonomic and geographic sampling support the monophyly of the marsupial frog genera (family Hemiphractidae), resolve six geographically circumscribed lineages within Gastrotheca, and, for the first time, reveal that two divergent lineages of Gastrotheca inhabit the Atlantic Coastal Forests of Brazil. Within Gastrotheca, the earliest diverging clade is confined to northeastern Brazil, whereas the three subsequent diverging lineages are restricted to northern Venezuela (G. walkeri), southeastern Brazil, and northwestern South America. All species in these clades inhabit humid forests at low to mid-elevations, and their life histories are characterized by lacking free-living tadpoles (i.e., direct development). Two derived clades inhabit the Andes, and both contain species with either direct development or tadpoles. One Andean clade of Gastrotheca ranges in the high Andes from Colombia to extreme northern Peru, whereas the other clade inhabits high elevations in the Andes of southern Ecuador, Peru, and Bolivia, and lower elevations in the Andes of northwestern Argentina. The presence of two non-sister lineages on each side of the Amazon Basin suggests that vicariance across this central region played an important role in diversification within Gastrotheca.