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 biodiversityandendemism, but the immigration processes of their unique floras are still poorly known. Here, we used Gesneriaceae as a proxy to investigate the immigration dynamics of tropical and subtropical Southeast Asian karst floras.We present the most comprehensive phylogenetic analysis of the Old World gesneriads to date based on twelve loci. By estimating divergence times and reconstructing ancestral states (habitat, soil type and range), we found that immigration into subtropical Southeast Asian karst floras first occurred in the Early Miocene, with two peaks in the Early–Middle Miocene and the Pliocene–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.We also discover that Southeast Asian karst biodiversity comprises 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. This study advances our understanding of the historical assembly of Southeast Asian karst floras.