The stomatal traits of 13 red and white grapevine cultivars of Vitis vinifera L. established in a cultivar collection garden in central Chile were characterized in this study. The cultivars examined were ‘Cabernet Franc’, ‘Cabernet Sauvignon’, ‘Carmenere’, ‘Cot’, ‘Grenache’, ‘Merlot’, ‘Mourvedre’, ‘Pinot Noir’, ‘Syrah’, ‘Chardonnay’, ‘Marzanne’, ‘Roussane’, and ‘Sauvignon Blanc’. Transparent nail polish peel prints in four adult leaves per vine were obtained and examined under a light microscope at 10 ×. The number of stomata in 0.196 mm2 was counted, and their length and width were measured using image analysis software. With these variables, the stomatal area and stomatal density were obtained. The ‘Cot’ cultivar had the largest stomata and stomatal area, while the ‘Mourvedre’ cultivar had the smallest stomata and stomatal area. ‘Chardonnay’ had the highest number of stomata and stomatal density, while ‘Carmenere’ had the lowest. The red grapevine cultivars exhibited larger and fewer stomata than the white grapevine cultivars, corroborating different adaptations for the environmental modulation of stomatal conductance and transpiration.