An extended survey showed that the Italian wild grapevine (Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi) population is still numerous, in spite of a range of threats. In this study, one hundred and sixty-one sites distributed in 10 Italian regions were explored and 814 wild samples were collected. The individuals were unequally distributed on all the national territory and they were mainly concentrated in some areas of Central and Southern Italy. The highest percentage of wild individuals was sampled in sites below 300 m of altitude with a connection to a hydrographic network, marginal from dense vegetation and with a high-quality environment conservation state. A possible correlation between the presence of wild grapevine individuals and carbonatic soil substratum was found. On 668 individuals, a genetic analysis was carried out by the 20 microsatellite loci commonly utilized for V. vinifera genotyping, to infer information on the population genetic diversity and structure; 645 individuals resulted as unique genetic profiles. According to Simple Sequence Repeat (SSR) analysis, the Italian wild population still conserves a high genetic diversity and a low inbreeding level. Moreover, any clear genetic structure was not observed inside the population, although a likely splitting between the two subpopulations from Central and Southern Italy was identified. Two alternative hypotheses were proposed to explain this result: (i) the beginning of a progressive differentiation process inside the population and (ii) the product of recolonization events occurred after the last glaciation.