The distribution of many vertebrate species in the Amazon basin is delimited by large rivers, which are frequently regarded as geographic barriers related with speciation and are therefore of high conservation value. Rapid development in the region includes plans to dam one of its thirteen major rivers, the Xingu. Environmental impact assessment is required for large scale development within the Brazilian portion of the Amazon Basin. However, impacts on biodiversity are only considered at the species level, and taxonomic work is far from described for many groups. In particular, anuran diversity is underestimated, in part owing to the scale of the region and, for some taxa, by morphological conservatism. Here we describe genetic and phenotypic variation in the brilliant-thighed frog, Allobates femoralis. We show that a unique genetic lineage, with a vocal repertoire distinct from that described for the species throughout its remaining geographic range, is located within the region to be directly and indirectly impacted by damming the Xingu River. Further, genetic variation within the A. femoralis group is largely structured in accordance to river systems, despite morphological conservatism. Our data add support for conservation policy to be amended to include intraspecific measures of diversity in order to more effectively conserve current biodiversity and evolutionary processes.