Diploid and tetraploid populations of the leptodactylid frog Odontophrynus americanus were studied in the Cordoba province (central Argentine) to evaluate whether they represent a cryptic species pair rather than forms of the same species as they are considered at present. We examined three independent character complexes (external morphology, advertisement calls, allozymes) and quantified the character-specific state of differentiation between diploids and tetraploids in comparison to that of the congeneric and widely sympatric O. occidentalis. Multivariate analysis of 15 morphometric characters indicated a remarkable degree of differention between both forms, but did not permit an unequivocal identification of individuals (rate of erroneous classification: 25%), whereas all O. occidentalis were classified correctly. In contrast, the temporal structure of the advertisement call permitted a distinction of diploids, tetraploids and O. occidentalis even based on a single call. The latter differed from the other by producing a call consisting of several pulse trains. The pulse rate of the O. americanus call, which consists of a single pulse train, was significantly lower in tetraploids than in diploids. The allozyme pattern and the corresponding allele frequencies indicated a close relationship among the three Odontophrynus taxa, but genetic distances obtained suggested a differentiation of gene pools at the level of species. Considering the degree of differentiation demonstrated in this study, the ploidy and the different geographical distribution, we conclude that diploids and tetraploids represent two species, similar to its North American counterpart Hyla chrysoscelis/H. versicolor. As the name O. americanus refers to tetraploids collected near Buenos Aires (Argentina), we describe the diploids as the new species Odontophrynus cordobae sp. nov.