Muscadine grape ( Vitis rotundifolia) is a popular fruit in the Southeastern United States because of its unique aroma and strong antioxidant capacity. Volatile compounds of a locally cultivated muscadine cultivar Cowart were characterized by solid-phase microextraction coupled with GC-MS. Twenty-eight volatile compounds, including fruity short-chain esters, alcohols, terpenes, and carbonyl compounds, were detected based on mass spectra and Kovats indices. Based on principal component analysis and hierarchical clustering, the grapes in stages I and II had relatively similar flavor patterns, which were different from that in stage III. Butyl-2-butenoate, hexyl acetate, propyl acetate, ethyl trans-2-butenoate, hexyl-2-butenoate, ethyl acetate, butyl acetate, 1-octanol, ethyl hexanoate, and β- citral were present as distinct volatile chemicals in stage III, while nonanal, decanal, and β-citronellol were distinct in stage II, and myrcenol, β-ocimene, and l-limonene were biomarkers in stage I. Understanding volatile compounds at each stage can assist farmers in choosing the optimal time to harvest muscadine grapes.