Cassava (Manihot esculenta subsp. esculenta) is a staple crop with great economic importance worldwide, yet its evolutionary and geographical origins have remained unresolved and controversial. We have investigated this crop's domestication in a phylogeographic study based on the single-copy nuclear gene glyceraldehyde 3-phosphate dehydrogenase (G3pdh). The G3pdh locus provides high levels of noncoding sequence variation in cassava and its wild relatives, with 28 haplotypes identified among 212 individuals (424 alleles) examined. These data represent one of the first uses of a single-copy nuclear gene in a plant phylogeographic study and yield several important insights into cassava's evolutionary origin: (i) cassava was likely domesticated from wild M. esculenta populations along the southern border of the Amazon basin; (ii) the crop does not seem to be derived from several progenitor species, as previously proposed; and (iii) cassava does not share haplotypes with Manihot pruinosa, a closely related, potentially hybridizing species. These findings provide the clearest picture to date on cassava's origin. When considered in a genealogical context, relationships among the G3pdh haplotypes are incongruent with taxonomic boundaries, both within M. esculenta and at the interspecific level; this incongruence is probably a result of lineage sorting among these recently diverged taxa. Although phylogeographic studies in animals have provided many new evolutionary insights, application of phylogeography in plants has been hampered by difficulty in obtaining phylogenetically informative intraspecific variation. This study demonstrates that single-copy nuclear genes can provide a useful source of informative variation in plants.