Muscadine grape (Vitis rotundifolia) is the first native North American grape to be domesticated. During the past century, breeding programs have created a large collection of muscadine cultivars. Muscadine cultivars are usually identified by evaluating morphological traits and checking breeding records, which can be ambiguous and unauthentic. During this study, simple sequence repeat (SSR) markers were used to generate DNA fingerprinting profiles to identify muscadine cultivars and verify their reported pedigrees. Eighty-nine Vitis accessions were genotyped using 20 SSRs from 13 linkage groups. From these, 81 unique subgenus Muscadinia accessions were identified, and a core set of five SSR markers was able to distinguish all of them. Eighteen misidentifications were found, and five previously unknown accessions were matched with cultivars in the dataset. The profiles of 12 cultivars were not consistent with their reported parentage–progeny relationships. Genetic diversity was analyzed at four levels: all V. rotundifolia cultivars (N = 67); current cultivars (N = 39); historical cultivars (N = 28); and wild V. rotundifolia accessions (N = 9). There was substantial genetic diversity in both wild and historically cultivated muscadines. The principle coordinate analysis (PCoA) showed clear separation among subgenus Vitis cultivars, wild muscadine accessions, and cultivated muscadines, with PCoA1 and PCoA2 explaining 11.0% and 9.3% of the total variation, respectively.