The origin of the outstanding Neotropical biodiversity is still debated. A comprehensive understanding is hindered by the lack of deep-time comparative data across wide phylogenetic and ecological contexts. Here we define and evaluate four evolutionary scenarios assuming different diversity trajectories and drivers of Neotropical diversification. Relying on 150 phylogenies (12,512 species) of seed plants and tetrapods, we found that diversity mostly expanded through time (70% of the clades), but scenarios of saturated (21%) and declining (9%) diversity also account for a substantial proportion of Neotropical diversity. These scenarios occur indistinctly across the major regions, habitats, and altitudes of the Neotropics, suggesting no geographic structure of Neotropical diversification. On the contrary, diversification dynamics differ across taxonomic groups: plant diversity mostly expanded through time (88%), while for a significant fraction of tetrapods (43%) diversity accumulated at a slower pace or declined toward the present. These opposite evolutionary patterns reflect different capacities for plants and tetrapods to cope with environmental change, especially in relation to climate cooling. Our results suggest that the assembly of Neotropical diversity is a long, clade-specific, and complex process resulting from a combination of gradual and pulse dynamics associated with environmental stability and instability over macroevolutionary scales.