Ex situ origins and dispersal of taxa have played important roles in the assembly of island-like biodiversity hotspots. Insular limestone karsts in Southeast Asia are hotspots of biodiversity and endemism, but the immigration processes of their unique floras are still poorly known. Here, we used Gesneriaceae as a proxy to investigate immigration dynamics of tropical and subtropical Southeast Asian karst floras. We show that immigration into subtropical Southeast Asian karst floras first occurred in the early Miocene, with two peaks in the early to middle Miocene and the Pliocene to early Pleistocene, whereas immigration into tropical Southeast Asian karsts initiated in the late Eocene, with two peaks in the late Oligocene and the late Miocene. The rich geological and ecological diversities of different regions, together with habitat isolation due to climatic changes, might be responsible for the different immigration patterns. We also discover that Southeast Asian karst biodiversity comprises a mix of immigrant pre-adapted lineages and descendants from local acid soil ancestors, although niche shift from acid soil to karst in tropical Southeast Asian islands was lacking. These findings provide insights into how Southeast Asian karsts respond to ongoing climate change and are of particular importance for their conservation planning.