Wild grapevine – Vitis vinifera subsp. sylvestris – ancestors of cultivated grapevines are the main players in understanding the molecular bases of the grapevine domestication process. The goal of the presented research was to assess the genetic diversity of wild grapevine samples from several regions encompassing Europe (Spain, France, Germany, Hungary, Greece), the Mediterranean basin (Algeria, Tunisia, Morocco), and South Caucasus (Georgia), using a complete chloroplast DNA sequencing. The results suggest the existence of three different chloroplast DNA haplotypes, reflecting the geographical distribution of the analyzed samples. This study represents the first report focused on analysis of a wide range of wild grapevine samples (Vitis vinifera subsp. sylvestris), applying next-generation technologies, and tracing the grapevine ancestry