Dioscorea rotundata Poir. is mainly dioecious but it also produces monoecious individuals. Recently two genetic markers were proposed to determine the sex in this species. We tested them in 119 individuals from 101 different cultivars of the national collection of Benin to verify whether they can predict the sex observed in the field. Among the analyzed individuals, 72 were male, 37 were female, 7 were monoecious and 3 were non-flowering. Our results showed that the marker sp16, associated with the W allele (female allele), was present in all female individuals but also in more than 42% of male individuals. Thus, while the absence of sp16 confirmed the male sex of the individuals, its presence did not allow sex discrimination. The marker sp1 allowed the identification of four genotypes (AA, AB, BB and AC) in the analyzed individuals with AA and AB being the most represented. Although AA was observed in 62.16% of female individuals and AB in 83.33% of male individuals, we did not observe a clear correlation between sp1 genotypes and sex identity. We concluded that the tested markers did not allow a clear sex discrimination in Beninese Dioscorea rotundata cultivars. Our results also suggest that Beninese D. rotundata cultivars have adopted a male XX/XY heterogametic system that is undergoing reorganization.