The complex geography of the Neotropical montane system is a natural laboratory for population divergence. Understanding which geographic barriers (lowland barriers, arid river valleys, and montane barriers above the tree line separate these regions of endemism) are instrumental in promoting and maintaining population divergence is an important step in preserving genetic diversity and endemism within the region. Here, I analyze patterns of genetic differentiation between 16 predefined regions of endemism for 43 co-distributed zoogeographic species complexes of Neotropical montane forest birds. The analysis shows that lowland barriers generate the highest levels of genetic differentiation, while barriers above the tree line in the Andes show the least. Within the Andes, arid river valleys promote population divergence to varying degrees. The Río Marañón shows the greatest effect, but the Río Apurímac and Río Quinimari are also associated with extensive genetic differentiation, while genetic divergence across other river valleys is generally weak. Most barriers are associated with a wide span of divergence times, supporting a protracted history of dispersal postdating barrier formation. If the goal is to maintain genetic diversity, preservation of populations within each region of endemism would help to ensure the continued survival of evolutionarily distinct lineages within species. Considering the alarming rate of deforestation in Neotropical montane regions, preservation of suitable tracts of montane forest is needed within each region of endemism, with special emphasis placed on endemism regions separated by lowland barriers and by deep intermontane river valleys.