A total of sixty-five accessions from the Spanish region of Castilla y León including those authorized in the VQPRD areas were characterized for six SSR loci. All the samples but one unknown were identified by comparison to other databases. Thirty synonymous samples and three cases of homonymy were found out, confirming in most cases ampelographic expectations. Five unique genotypes belonging to local varieties in risk of extinction were detected. Several parameters were calculated to assess the usefulness of the chosen loci in this work. A dendrogram representing the genetic similarities among the accessions was constructed using the neighbor-joining method to investigate possible parentage relationships in the sample, and to explain them from an historical and cultural point of view.