The genetic relationships of twenty seedless grape cultivars, used as parents for seedless grape breeding, were analyzed and the validation assay of the P3_VvAGL11 marker related to the seedlessness was examined. Total 218 polymorphic bands were generated by 30 SSR markers. The average number of alleles per marker was 7.3. The polymorphism information contents (PIC) averaged 0.442, value ranging from 0.052 to 0.083 by each SSR marker. Eighteen seedless grape cultivars derived from ‘Sultanina’ and ‘Kishmish Chernyi’ had 196-bp alleles of the P3_VvAGL11 associated with seedlessness. The unweighted pair-group method arithmetic average (UPGMA) cluster analysis was performed using selected 218 polymorphic bands. And four groups were derived on the similarity index 0.722. Each group accorded with the classification by the ratio of the European species (Vitis vinifera), the American species (V. labrusca) and the inter-specific hybrid between the two species (V. labruscana). Similarity values among the tested seedless grape cultivars were ranged from 0.592 for ‘Beauty Seedless’ and ‘Honey Seedless’ to 0.844 for ‘Emerald Seedless’ and ‘Ruby Seedless’, the average similarity value was 0.715. These results could be used as useful reference for selecting parents with diverse backgrounds and early selection of seedless seedlings in breeding program of seedless grape cultivars.