The composition and content of polyphenols in the seeds of 91 grape accessions from 17 Vitis species were characterized. Eleven compounds, including 2 gallic derivatives, 3 monomeric flavan-3-ols, 3 flavonols, resveratrol, and procyanidin B1 and B2, were identified via HPLC-MS and quantified by HPLC-DAD. In addition, seventeen dimeric and trimeric flavan-3-ols were also quantified. Tremendous variation was observed both among and within species for these compounds. Monomeric flavan-3-ols were the most abundant polyphenols in seeds, followed by dimeric and trimeric flavan-3-ols, which collectively accounted for more than 96% of the total polyphenols. V. palmata, V. vinifera, and V. vulpina had significantly higher content of total polyphenols than other species. A number of Vitis accessions with high content of various types of seed polyphenols were identified, and they can serve as potential germplasm for improving the composition and content of seed polyphenols in cultivated grapes.