The North American muscadine grape (Museadinia rotundifolia Small) is a valuable source of resistance to powdery mildew [Uncinula necator (Schw.) Burr], root-knot nematode (Meloidogyne Goeldi), dagger nematode (Xiphinema index Thorne and Allen), grape phylloxera (Daktulosphaira vitifoliae Fitch), and Pierce's disease (Xylella fastidiosa Wells et al.). Efforts to breed muscadine grapes commenced in the early 1900s and have generated a large number of cultivars and a limited number of hybrids with Vitis vinifera L. and other Vitis L. species. Collections of this germplasm are currently maintained with accession identity based on declared identity when collected, breeding records, and comparisons of morphological traits. This study reports on the first use of DNA-based simple sequence repeat (SSR) marker profiles to authenticate M. rotundifolia cultivars and hybrids. A total of 57 accessions [39 M. rotundifolia cultivars, 3 V. vinifera cultivars, 3 Vitis spp. hybrids, and 12 V. vinifera X M. rotundifolia (VR) hybrids] from collections at the U.S. Department of Agriculture National Clonal Germplasm Repository and the University of California (Davis) Department of Viticulture and Enology were analyzed with 14 SSR markers. The fingerprint profiles were used to verify published breeding records of 31 M. rotund folia cultivars and hybrids by comparing the shared alleles of parents and progeny. Marker data indicated that four cultivars were incorrectly identified; their alleles did not match respective parent/progeny relationships at more than five loci. Two M. rotundifolia accessions had the same fingerprint profile as a third accession at all 14 markers, implicating a likely planting error. The M. rotundifolia cultivars exhibited 88 unique alleles that were not present in a database of more than 600 V. vinifera cultivars.