We present a new phylogeny, based on DNA sequences of mitochondrial and nuclear genes, for frogs of the genus Osteocephalus with emphasis in the Osteocephalus buckleyi species complex. Genetic, morphologic, and advertisement call data are combined to define species boundaries and describe new species. The phylogeny shows strong support for: (1) a basal position of O. taurinus + O. oophagus, (2) a clade containing phytotelmata breeding species, and (3) a clade that corresponds to the O. buckleyi species complex. Our results document a large proportion of hidden diversity within a set of populations that were previously treated as a single, widely distributed species, O. buckleyi. Individuals assignable to O. buckleyi formed a paraphyletic group relative to O. verruciger and O. cabrerai and contained four species, one of which is O. buckleyi sensu stricto and three are new. Two of the new species are shared between Ecuador and Peru (O. vilmae sp. n. and O. cannatellai sp. n.) and one is distributed in the Amazon region of southern Peru (O. germani sp. n.). We discuss the difficulties of using morphological characters to define species boundaries and propose a hypothesis to explain them. © Santiago R. Ron et al.