For decades, human activities have gradually destroyed the natural habitats of wild grapevine, Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi, and nowadays this species is endangered in southern Europe. In this paper, 94 populations of this species have been localized and characterized in the Andalusian region in the Iberian Peninsula between 1989 and 2013. Location, ecological aspects, and sanitary characteristics are described. Must properties and in vitro tolerance to calcareous conditions were also checked. The paper also contains a global description of female and male individuals. Two hundred individuals from six river basin populations have been sampled, and their genetic structure analyzed by using 25 nuclear microsatellites loci to investigate the gene diversity of wild grape populations in Andalusia at two levels: total individuals and at river basin populations. Also, the genetic relationship of wild and cultivated accessions has been tested. Wild grapevine is considered the ancestor of the cultivated varieties and should be preserved as this material could be used to start breeding programs of cultivated varieties and also to restore riverbank forests, which constitute one of the worst preserved ecosystems in the area.