While a growing body of modern phylogenetic research reveals that the Western indochina represents a separate biogeographic subregion having a largely endemic freshwater fauna, the boundaries of this subregion are still unclear. We use freshwater mussels (Unionidae) as a model to reconstruct spatial patterns of freshwater biogeographic divides throughout Asia. Here, we present an updated freshwater biogeographic division of mainland Southeast Asia and describe 12 species and 4 genera of freshwater mussels new to science. We show that the Isthmus of Kra represents a significant southern biogeographic barrier between freshwater mussel faunas of the Western indochina and Sundaland subregions, while the Indian and Western Indochina subregions are separated by the Naga Hills, Chin Hills, and Rakhine Yoma mountain ranges. Our findings highlight that the freshwater bivalve fauna of Southeast Asia primarily originated within three evolutionary hotspots (Western Indochina, Sundaland, and east Asian) supplemented by ancient immigrants from the indian Subcontinent. Freshwater mussels (Unionida) are an economically and environmentally important group of aquatic animals having a broad distribution on all continents except Antarctica 1,2. Southeast Asia houses one of the richest endemic faunas of freshwater mussels globally 3-7. Unfortunately, freshwater mussels are among the most endangered animal groups at the global scale, with numerous local extinctions triggered by multiple anthropogenic impacts and climate changes 8-11. Human-mediated degradation of natural habitats, e.g. water pollution, river damming, and irrigation practices, appears to be the most influential factor causing the decline and local extinctions of freshwater mussels 11-13. It was shown that even a prehistoric decline in freshwater mussels corresponds to the early development of agricultural techniques 14. Alien species may represent a significant threat to native freshwater mussel assemblages in Southeast Asia 13 and other regions 15. For example, the tropical lineage of Sinanodonta woodiana (Lea, 1834) is widely spread throughout Malaysia, the Indonesian Archipelago, and the Philippines 16-18 , while the temperate lineage of this taxon was found in Myanmar 19. Recent advances in mitogenomic 20 and multi-locus nuclear 21 phylogenetic modeling reveal that two widespread Southeast Asian subfamilies of the Unionidae, i.e. Pseudodontinae 22 and Rectidentinae 22,23 , represent tribes within the monophyletic Gonideinae. The genus-and species-level taxonomy of freshwater mussels in Southeast Asia is still poorly known 3 , but several integrative studies performed in Myanmar, Thailand, Laos, and Malaysia have recently improved our knowledge about the diversity and biogeographic patterns in the region.