Background: Greater yam (Dioscorea alata L.) is a major tropical and subtropical staple crop cultivated for its starchy tubers. Breeding of this dioecious species is hampered by its erratic flowering, yet little is currently known on the genetic determinism of its sexual reproduction. Result: Here we used a genome-wide association approach and identified a major genetic barrier to reproduction in yam on chromosome 1, as represented by two candidate genes. A deleterious effect on male fitness could be hypothesized considering the involvement of these two genes in male reproduction and the low frequency of this non-flowering dominant allele within the male genepool. We also extended the hypothesis of a XX/XY sex-determination system located on chromosome 6 in D. alata to encompass most of the species diversity. Moreover, a kompetitive allele-specific PCR (KASPar) marker was designed and validated that enables accurate cultivar sex estimation. The reconstruction of chromosome 6 associated with the detection of highly putative structural variations confirmed the possible involvement of a major part of the chromosome. Conclusion: The findings of this study, combined with proper estimation of accession ploidy levels to avoid endosperm incompatibility issues, could facilitate the design of future promising parental combinations in D. alata breeding programs. Moreover, the discovery of this genetic barrier to reproduction opens new avenues for gaining insight into yam reproductive biology and diversification.