Abstract Neotropical diversification by the Andean uplift is typically addressed on a large evolutionary scale ( e . g ., speciation), even though many species are still distributed in both sides of the mountains. The three parallel mountain ranges in the northern Andes (Colombia) impose a major geographical barrier to species’ migration from South to Central America. How important these barriers are for conspecific diversification of cross‐Andean species such as the leaf‐cutting ants remains largely unknown. To answer this question, we studied the mtCOI gene of Atta cephalotes , the most widely distributed leaf‐cutting ant species. Our hierarchical analyses evidenced substantial genetic structure among regions and populations, suggesting a more complex biogeographical history of Andean populations than previously thought. These mountains seem to isolate Central American and Western Colombian populations from the rest of A . cephalotes in South America. Population and migration modeling are consistent with the origin of this species in South America and a major role of the Eastern cordillera as a geographical barrier to historical gene flow, restricting dispersion from north to south. These findings provide insights into the role of the Andean uplift as barrier to gene flow in species of leaf‐cutting ants. Abstract in Spanish is available with online material.