Tropical Southeast Asia harbors extraordinary species richness and in its entirety comprises four of the Earth's 34 biodiversity hotspots. Here, we examine the assembly of the Southeast Asian biota through time and space. We conduct meta-analyses of geological, climatic and biological (including 61 phylogenetic) datasets to test which areas have been the sources of long-term biological diversity in SE Asia, particularly in the pre-Miocene, Miocene and Plio-Pleistocene, and whether the respective biota have been dominated by in situ diversification, immigration and/or emigration, or equilibrium dynamics. We identify Borneo and Indochina, in particular, as major ‘evolutionary hotspots’ for a diverse range of fauna and flora. While most of the region's biodiversity is a result of both the accumulation of immigrants and in situ diversification, within-area diversification and subsequent emigration have been the predominant signals characterizing Indochina and Borneo's biota since at least the early Miocene. In contrast, colonization events are comparatively rare from younger volcanically active emergent islands such as Java, which show increased levels of immigration events. Few dispersal events were observed across the major biogeographic barrier of Wallace's Line. Accelerated efforts to conserve Borneo's flora and fauna in particular, currently housing the highest levels of Southeast Asian plant and mammal species richness, are critically required.