Southern Amazonia potentially harbors a wide diversity of aquatic macrophyte species because of its diverse wetland habitats and location in the Amazon-Cerrado transition zone, the two largest biogeographic domains in South America. We inventoried the taxonomic diversity of aquatic macrophytes in the region, including endemism, habitats, species life forms, and floristic comparisons with other Brazilian regions. We found a high number of species of aquatic macrophytes for the southern Amazon region, comparable to extensive inventories in larger areas in southern, northeastern, and northern regions of Brazil. We recorded 688 species of aquatic macrophytes in 299 genera and 95 families, of which 66 species are new records, 89 endemic, and 5 exclusive to Brazilian Amazonia. We present new records of aquatic macrophyte species, together with their degree of endemism and occurrence restricted to the region. The macrophyte species list of southern Amazonia showed 60% similarity to inventories in the Xingu basin and in northern Brazil. This high diversity of aquatic macrophytes in the Southern Amazonia, with endemic species and others with restricted ranges, emphasizes the importance of conserving wetlands and vegetation types where they grow in the region.