Vitis vinifera ssp. vinifera (domesticated grapevine) includes thousands of cultivars, which are classified according to their main uses, as wines, fresh fruits or dried raisins and sultanas since ancient times. Evidence showed that Crete grapevine cultivars and winemaking date back to 2300 BC. In this study, fifty-one genotypes belonging to seven different traditional Vitis vinifera cultivars, presumed autochthonous to the island of Crete, were selected for their wine-producing potential and classified by 51 ampelographic descriptors. In addition, five genotypes belonging to two non-autochthonous cultivars were included as out-group controls. Subsequently, in order to characterize genetic diversity, establish genetic relationships within and between cultivars and solve accession-labeling problems, genotypes were fingerprinted employing Simple Sequence Repeat (SSR or microsatellite) markers. Four of the autochthonous cultivars namely ‘Vidiano’, ‘Vilana’, ‘Plyto’, and ‘Moschato Spinas’ are used in the local economy for blanc (white) wine production while the rest, namely ‘Kotsifali’, ‘Liatiko’ and ‘Mantilari’ for Noir (red) wines. The two cultivars employed as out-group were ‘Moschato Samou’ and ‘Moschato Alexandrias’: both white wine producers. Ampelography-based clustering grouped the majority of genotypes along cultivar-specific clusters. All three Moschato cultivars formed a distinct clade pointing to the non-autochthonous origin of ‘Moschato Spinas’. A total of one hundred and thirteen (113) SSR alleles were amplified from thirteen (13) SSR loci, with an average number of alleles per locus equal to 10.23 revealing ample genetic polymorphism. The cumulative probability of identity was also quite high (3.389 × 10−16). The overall observed heterozygosity was 0.837 while for twenty-nine of the examined genotypes, at least one private SSR allele was detected. The majority of genotypes were grouped in cultivar-specific clusters. The results of this paper pave the way for the certification and registration of clones of some of the most important wine-producing cultivars in Crete.