Chamberlainia hainesiana (Lea, 1856) (Unionidae) is the most economically important freshwater mussel in Thailand and is commonly used in food, spiritual ceremonies and pearl culture. Despite the clear economic importance of this monotypic genus, the distribution and diversity of Chamberlainia Simpson, 1900 are poorly understood. We set out to re-evaluate the taxonomic and geographic boundaries of Chamberlainia using molecular and morphological data gathered from recently and historically collected material. Our cytochrome c oxidase subunit I gene tree recovered three divergent species-level lineages in the genus Chamberlainia. Fourier shape analysis of 60 digitized Chamberlainia shell outlines revealed evidence of two morphologically divergent groups that could be statistically distinguished 88% of the time. We used this evidence to recognize two genetically and morphologically divergent Chamberlainia species, C. hainesiana and C. duclerci (Rochebrune, 1882) stat. res. Geographical information from 46 museum records was used to map the distribution of Chamberlainia. These results are discussed in the context of their taxonomic, biogeographic and conservation implications.