Yams (Dioscorea spp.) are one of the main root and tuber crops in the world, especially within the species complex Dioscorea cayenensis/D. rotundata. Few studies have been conducted in Brazil with these species, including genetic diversity. The objective of this study was to characterize the genetic diversity of local varieties of D. cayenensis and D. rotundata using morphological and molecular markers, and provide information on the management and use of the crop by family farmers from different regions in Brazil. Thus, yam tubers were sampled from several municipalities in the South, Southeast and Northeast regions. Eighteen morphological traits and ten microsatellite loci were used to analyse 47 yam accessions (23 D. cayenensis and 24 D. rotundata). Species identification was carried out after field morphological evaluation. Spatial genetic analysis indicated significant structure among the local varieties, mostly between regions and species. Both cluster and Bayesian analyses showed a separation of the accessions into two distinct groups: group I with accessions originated from the Southeast region and group II with accessions originated from the Northeast region, while accessions from the South region were intermediate or included in either group. The results showed a separation between D. cayenensis and D. rotundata accessions in Brazil, and that D. cayenensis occurs predominantly in the Southeast region, while D. rotundata occurs in the Northeast region. Further studies with larger sampling would be welcome in order to confirm these findings. Also, this study highlights the importance of family farmers in the genetic diversity conservation of these species in Brazil.