The fundamental question about Dioscorea trifida (Dioscoreaceae), the most important Amerindian yam, that remains unresolved concerns its evolutionary origin, since no wild relative has been reported. In this paper we report the existence of D. trifida's wild relative for the first time. The diploidy of wild D. trifida (2n = 40) is clearly demonstrated by flow cytometry, chromosome counts, and microsatellite pattern analysis, whereas the cultivated form was previously shown to be autotetraploid (2n = 80). In the coastal region where the wild and cultivated forms are sympatric, tetraploid and triploid cytotypes coexist within the same populations. In the sites where the wild and cultivated forms are allopatric, the wild diploid cytotype predominates. AFLP (amplified fragment length polymorphism) analyses gave an initial idea of the position of the wild forms in relation to the cultivated forms. All the wild and cultivated types form a monophyletic group structured into two major subgroups corresponding to the tetraploid cytotype of the cultivated form and the diploid cytotype of the wild form. The triploid cytotypes of the wild form are in an intermediary position. Wild accessions are grouped on the basis of their geographic origin. The data presented in this paper are significant for the effective breeding and conservation of D. trifida and to assess its genetic diversity and population structure for the general understanding of the evolution and domestication of the species.