Despite the economic and cultural importance of the indigenous "Amerindian" yam Dioscorea trifida, very little is known about their origin, phylogeny, diversity and genetics. Consequently, conventional breeding efforts for the selection of D. trifida genotypes resistant to potyviruses which are directly involved in the regression of this species have been seriously limited. Our objective of this paper is to contribute to the clarification of the cytogenetic status, i.e., inheritance and chromosome number. Our results provide genetic evidence supporting tetrasomic behaviour of the genome of D. trifida based on chromosomal segregation pattern analysis using eight SSRs markers in three different crosses. This is the first reliable evidence of an autopolyploid species in the genus Dioscorea. The second major result in this study is the revealing of a new base chromosome number in the botanical section Macrogynodium to which D. trifida belongs. To date, our assumptions about the ploidy level of yams are based on the observations that the basic chromosome number is 10 or 9, and D. trifida was described as octoploid. The chromosome number of D. trifida accessions was also assessed using somatic chromosomic count techniques. Flow cytometry did not show significant variation of 2C DNA content among 80 accessions indicating homogeneity of the ploidy level of the cultivated D. trifida. This suggests that autotetraploidy is well established as well as the rule for the cultivated pool of D. trifida, even if the direct diploid ancestor remains to be identified. The data presented in this paper are significant and important for the effective breeding and conservation of the species and for elucidating the phylogeny and the origins of the yam and the evolution of the genus Dioscorea.