Among frogs endemic to the Colombian Andes, the dendrobatid Colostethus palmatus has an exceptionally wide geographic distribution. We examined the patterns of variation in calls and genetic properties to determine whether this variation is associated with geographic barriers and distances between populations. We analyzed variation in advertisement calls and RAPD products in seven populations along a 346-km transect from south to north throughout the Cordillera Oriental covering an extensive part of the geographical range of C. palmatus. Populations of this species inhabit both slopes of the Cordillera Oriental, and are separated by a continuous ridge along these mountains. We found more inter- than intrapopulational variation. Four call variables and male snout–vent length exhibited clinal variation, generating a predictable geographic pattern which coincided with a climatic gradient of increasing moisture and seasonality. The genetic relations between populations are explained by their location on the Andes, particularly by the side of the slope. Differences between populations in four call characteristics and genetic distance were significantly correlated with linear geographic distances between populations on the same slope. We found evidence for genetic and bioacoustic differentiation between populations living on opposite slopes.