Physical paleoaltimetric methods are increasingly used to estimate the amount and timing of surface uplift in orogens. Because the rise of mountains creates new ecosystems and triggers evolutionary changes, biological data may also be used to assess the development and timing of regional surface uplift. Here we apply this idea to the Peruvian Andes through a molecular phylogeographic and phylochronologic analysis of Globodera pallida, a potato parasite nematode that requires cool temperatures and thus thrives above 2.0–2.5 km in these tropical highlands. The Peruvian populations of this species exhibit a clear evolutionary pattern with deeper, more ancient lineages occurring in Andean southern Peru and shallower, younger lineages occurring progressively northwards. Genetically diverging G. pallida populations thus progressively colonized highland areas as these were expanding northwards, demonstrating that altitude in the Peruvian Andes was acquired longitudinally from south to north, i.e. in the direction of decreasing orogenic volume. This phylogeographic structure is recognized in other, independent highland biotaxa, and point to the Central Andean Orocline (CAO) as the region where high altitudes first emerged. Moreover, molecular clocks relative to Andean taxa, including the potato–tomato group, consistently estimate that altitudes high enough to induce biotic radiation were first acquired in the Early Miocene. After calibration by geological and biological tie-points and intervals, the phylogeny of G. pallida is used as a molecular clock, which estimates that the 2.0–2.5 km threshold elevation range was reached in the Early Miocene in southernmost Peru, in the Middle and Late Miocene in the Abancay segment (NW southern Peru), and from the latest Miocene in central and northern Peru. Although uncertainties attached to phylochronologic ages are significantly larger than those derived from geochronological methods, these results are fairly consistent with coeval geological phenomena along the Peruvian Andes. They strongly suggest that orogenic volume initially developed in the CAO during most of the Miocene until a breakthrough in the latest Miocene allowed the northward propagation of crustal thickening into central and northern Peru, possibly by ductile crustal flow from the CAO. Such a combined phylogeographic and phylochronologic approach to regional uplift opens perspectives to estimate the direction(s) and timing of acquisition of altitude over other Cenozoic orogens.