We have used nuclear and chloroplast molecular markers to genotype cultivated and wild accessions of Vitis vinifera L. from Tunisia and assess their genetic relationships. Fifty-five distinct genotypes were identified among 80 cultivated accessions, including 18 genotypic groups containing between 2 and 5 accessions per group. They could represent a total of 60 distinct cultivars owing to berry colour variation found within identical genotype groups. Most of the 55 genotypes represent unique table grape genotypes except for one of them that was found identical to the genotype of table grape cultivar Rosseti. Hybridization among cultivars as well as self pollinations seems to have played an important role in their origin since several groups of closely related cultivars were observed. Furthermore, a parentage analysis showed a high probability for a parent hybrid relationship within two groups of three cultivars. No strong genetic similarities were found between cultivated and wild samples indicating that the cultivated accessions do not derive from local Vitis vinifera L. populations but could have been introduced from other regions in historic times.