Guinea yam (Dioscorea rotundata Poir.) is a dioecious vegetatively propagated tuber crop. It is widely cultivated by traditional techniques in West Africa, its area of origin. The genetic diversity of 146 accessions from Benin was analysed using 10 polymorphic simple sequence repeat (SSR) nuclear markers and agromorphological traits. An average of 8.4 alleles per locus was detected. The mean heterozygosity was 0.57 and the mean polymorphism information content (PIC) for polymorphic markers was 0.51. Some cultivars (23%) were found to have an identical genotype for the 10 markers. The structure of the genetic diversity observed in Benin is the result of farmers' crop management practices and their know-how. The cultivar diversity had a geographical component. We also noted major differentiation between early and late cultivars, with higher diversity in the early ones. Cultivars from northern Benin and early cultivars had the greatest allelic richness. SSR markers proved to be powerful tools for fingerprinting each cultivar and analysing their genetic relationships. The results of this study could be useful for defining a strategy for the conservation of genetic diversity in yams.