There are two main competing hypotheses (vicariance and vertical ecotones) that attempt to explain the tremendous diversity of the tropical Andes. We test these hypotheses at the intraspecific level by analyzing mitochondrial and nuclear DNA sequences from 24 populations of the high Andean frog, Dendropsophus labialis (Anura: Hylidae). This species displays geographic variation in a number of phenotypic traits. Most of these traits covary with elevation, while few vary along the horizontal (latitudinal) axis. We found that, both, vicariance and elevation had important effects on the genetic differentiation in this species. We detected two highly divergent clades along the south–north axis using independent information from mitochondrial and nuclear genes, suggesting that this differentiation was the result of long-term barriers to gene flow rather than stochastic processes. We hypothesize mechanisms for D. labialis strong differentiation in light of geological and paleoenvironmental models of evolution in the northern Andean highlands.