Grape (Vitis vinifera L.) is among the most important commercial fruit species grown worldwide in terms of its economic value. Anatolia (Turkey), located in the favorable climate zone for viticulture, has a rich grape genetic potential due to its location at the intersection of the grapevine gene centers. In Turkish Kara grape germplasm, there are problems in terms of accuracy during the production phase due to the inadequacies in ampelographic definitions, and also very little information is available on genetic analysis of Kara grape germplasm. This study carried out genetic analysis of 49 Kara grape cultivars from six regions (sub-populations) of Turkey and 3 reference cultivars using 22 microsatellite loci (SSR), and ampelographic analysis were also performed concerning 39 OIV descriptors. In the SSR analysis, the average number of alleles per locus was 8.91, ranging from 4 to 13; four synonymous and five homonymous cases were also identified. In the population structure analysis, the genetic differentiation (Fst) values among six populations were moderate. In the BAPS analysis, all populations except Central Anatolia were found to be highly admixed with each other, and in the FCA analysis, the East Anatolia population was completely separated. In the multilocus lineages (MLLs) analysis, a total of three accessions were matched to different accessions as clone assignment. In this study, SSR-based genetic characterization of the Turkish Kara grape germplasm was revealed for the first time, and it is thought that the obtained data will help other grape genetic characterization studies and contribute to viticulture research in other areas such as breeding, protection and variety registration.