Late Pliocene and Pleistocene climatic instability has been invoked to explain the buildup of Neotropical biodiversity, although other theories date Neotropical diversification to earlier periods. If these climatic fluctuations drove Neotropical diversification, then a large proportion of species should date to this period and faunas should exhibit accelerated rates of speciation. However, the unique role of recent climatic fluctuations in promoting diversification could be rejected if late Pliocene and Pleistocene rates declined. To test these temporal predictions, dateable molecular phylogenies for 27 avian taxa were used to contrast the timing and rates of diversification in lowland and highland Neotropical faunas. Trends in diversification rates were analyzed in two ways. First, rates within taxa were analyzed for increasing or decreasing speciation rates through time. There was a significant trend within lowland taxa towards decreasing speciation rates, but no significant trend was observed within most highland taxa. Second, fauna wide diversification rates through time were estimated during one-million-year intervals by combining rates across taxa. In the lowlands, rates were highest during the late Miocene and then decreased towards the present. The decline in rates observed both within taxa and for the fauna as a whole probably resulted from density dependent cladogenesis. In the highlands, faunawide rates did not vary greatly before the Pleistocene but did increase significantly during the last one million years of the Pleistocene following the onset of severe glacial cycles in the Andes. These contrasting patterns of species accumulation suggest that lowland and highland regions were affected differently by recent climatic fluctuations. Evidently, habitat alterations associated with global climate change were not enough to promote an increase in the rate of diversification in lowland faunas. In contrast, direct fragmentation of habitats by glaciers and severe altitudinal migration of montane vegetation zones during climatic cycles may have resulted in the late Pleistocene increase in highland diversification rates. This increase resulted in a fauna with one third of its species dating to the last one million years.