Wild grapevine Vitis vinifera ssp. sylvestris species are endangered in their natural habitats by modern landscape use and thus there is need of further collection and preservation of these species in ex situ collections or the preservation in situ. In this study 34 wild Vitis vinifera ssp. sylvestris accessions from the Upper Rhine Valley including a defined subpopulation are described and compared with six accessions from the former Yugoslavia stored in an ex situ collection. The accessions are described by means of ampelographic descriptors and genotyped at six SSR loci. Both marker types were helpful to characterize the V. vinifera ssp. sylvestris ex situ collection. Differentiation in accession groups was found based on genotypic variation. Ampelographic traits split the accession in two major groups, where one group holds mostly accessions from the Ketsch area and the Upper Rhine Valley. Preservation of V. vinifera ssp. sylvestris genotypes is essential for the maintenance of genetic diversity and the resistance of genetic erosion. More accessions of this species should be collected and conserved for conversation and future breeding applications.