The wild grapevine, Vitis vinifera L. ssp sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain natural populations of Vitis vinifera ssp sylvestris can still be found along river banks. In this work we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analyzed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated and wild germplasm are genetically divergent with low level of introgression. Using a model-based approach implemented in the software STRUCTURE we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships among wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current western cultivars.