Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%-88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.