Grapevine (Vitis vinifera L.) is a major worldwide crop of high economic importance, tightly interwoven with the traditions and the culture of many civilizations. The Greek vineyard is one of the oldest in the world composed of an ample number of highly diverse indigenous landraces. However, over the last decades the local cultivated grapevine germplasm has undergone a drastic reduction of diversity, due to the established market preferences for international varieties. In the current work a combined approach involving both, ampelographic traits and microsatellite markers has been undertaken, to study the genetic diversity within and among 96 grapevine genotypes belonging to 36 V. vinifera subsp. vinifera cultivars, predominantly representing autochthonous Greek landraces. Results revealed high genetic diversity for the Greek cultivars yielding a mean number of alleles per locus 14.69 and mean polymorphic information content 0.848. Hierarchical cluster analysis, employing both, ampelographic and microsatellite data, showed a clear distinction based on the origin of the germplasm; Anatolian versus Mediterranean. Principal component analysis, based on the most informative ampelographic traits, coupled to the results from genetic structure analysis further corroborated the proposal of germplasm differentiation on the basis of geographic origin. This information can be further utilized for the reconstitution of the Greek vineyard and can significantly contribute towards a rational conservation and utilization strategy for breeding or for direct cultivation of the Greek indigenous grapevine germplasm.