The assembly of the world’s most biodiverse region, the Neotropics, was traditionally considered the result of long-term environmental stability. Studies gathered during the last decades suggest that environmental “instability” instead, specially the dramatic modifications caused by the uplift of the Andes, was responsible of the Neotropical diversity. Yet a comprehensive understanding has been hindered by a lack of large-scale comparative data across wide phylogenetic and ecological contexts. Here, we evaluate the timing and drivers of Neotropical diversification in a large sample of Neotropical clades: 150 phylogenies (12,524 species) of seed plants and major tetrapods (amphibians, mammals, squamates, and birds). We unveil five trends: (1) biodiversity levels before the Quaternary were comparable (or higher) to those of the present,; (2) half of the clades diversified at constant rates; (3) past environmental variations correlate with diversification changes in 37% of the lineages, but with contrasting responses: (4) birds and mammals diversified extensively during warm periods and global cooling resulted in synchronized slowdowns of diversification; plant diversification generally increased during cooling; and (5) the rise of the Andes mostly impacted amphibians and squamates. Our study suggests that environmental instability over macroevolutionary scales may in fact act as a driving force of Neotropical diversification.