AbstractHighlands are of paramount importance to the study of evolution as they are frequently implicated in historical and ecological processes that generate and maintain biological diversity. In northeastern Brazil, sparse rainforest remnants located in highlands north of the São Francisco River are surrounded by the dry and open landscape of the Caatinga biome. Earlier studies suggested that these forests were historical refuges to the rainforest fauna and flora during Pleistocene's climatic cycles. However, it is still unclear whether populations in distinct highlands experienced phenotypic differentiation as a result of adaptation to environmental conditions of each forest remnant. Herein, we used two frog species widely distributed and ecologically different, Dendropsophus oliveirai, a habitat specialist, and Physalaemus cuvieri, a habitat generalist, to investigate the relationships between environmental variation, geographic, genetic, and body size distance with advertisement call variation among populations inhabiting different highlands. Our results indicated that acoustic variation among P. cuvieri populations is strongly influenced by environmental variation, but also by the geographic distance among populations. In D. oliveirai, environment is also the most influent factor on acoustic variation, followed by a lower influence of genetic and morphological variation. Associations between environmental and geographic factors suggest indirect effects of geographic distance on acoustic variation in both species through an environmental gradient. We believe that selective processes and isolation by distance possibly act together in driving interpopulational acoustic variation with habitat‐specific species being more affected by geographic isolation in suitable habitats.Abstract in Portuguese is available with online material.