As the ancestor of cultivated grape, Vitis vinifera subsp. sylvestris represents a unique, invaluable genetic resource for the improvement of cultivated grapevines. Recently, five populations of wild grapevines were identified in Portugal. Sixty vines were characterized with 11 nuclear SSR markers and further compared with 70 genotypes of Portuguese Vitis vinifera subsp. sativa. The obtained data demonstrate moderate genetic differentiation between wild grapevine populations and moderate to high genetic differentiation between wild and cultivated grapevines. However, the identification of high degrees of similarity between wild and cultivated grapes (up to 87%) and a putative parent-progeny relationship between wild and cultivated grapes with 17 additional SSR markers is indicative of gene flow between local wild grapevine populations and Portuguese domesticated vines. Also, the ancestry of some Azorean cultivars was ascertained. The obtained data further support the hypothesis of several domestication centres, with Portugal, Spain, and Italy playing a particular role after the last glaciation, giving rise to many of the Western European cultivars.