Muscadine grapes (Vitis rotundifolia Michx) are considered as excellent genetic resources for grape breeding programs as they are known for their hardiness and resistance to pests and diseases. However, contrary to popular belief, our study indicated that not all muscadine cultivars are resistant to anthracnose disease. In order to identify a source of genetic tolerance towards anthracnose among muscadine cultivars, a series of in-situ and ex-situ experiments were conducted through strict and sensitive screening processes. Two consecutive years of field evaluation of 54 grape cultivars showed various levels of anthracnose incidence among the cultivars between a scale of 0 (tolerant) to 5 (highly-susceptible). Resistance bioassay by inoculation of different spore densities of Elsinoe ampelina on 40 cultivars presented similar results and was consistent with those obtained from the field test. A real-time PCR analysis was conducted to investigate differences of gene expression between susceptible and tolerant cultivars and to confirm results by phenotypic identification. Expression of genes encoding chalcone synthase, stilbene synthase, polygalacturonase-inhibiting protein, chitinase and lipid transfer-protein was only detected in tolerant cultivars. Resistant muscadine cultivars identified in this study could be excellent candidates for grape disease resistance breeding programs.