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Global warming will significantly affect grapevine growth and development. To analyze the effects of high temperature on the leaf tissue structure of grapevines in the field, 19 representative cultivars were selected from the grapevine germplasm resources garden in Turpan Research Institute of Agricultural Sciences, XAAS. Twelve tissue structure indexes of grapevine leaves, including the thickness of the upper epidermis (TUE), the thickness of palisade tissue (TPT), leaf vein (LV), the thickness of spongy tissue (TST), the thickness of the lower epidermis (TLE), stoma (St), guard cell (GC), cuticle (Cu), leaf tissue compactness (CTR) and leaf tissue porosity (SR), were measured during the natural high-temperature period in Turpan. The results showed significant differences in the leaf tissue structure of the 19 grapevine cultivars under natural high temperature. Based on the comprehensive comparative analysis of the leaf phenotype in the field, we identified that the leaves of some cultivars, including ‘Zaoxia Wuhe’, ‘Centennial Seedless’ and ‘Kyoho’ showed strong heat tolerance, whereas grapevine cultivars ‘Golden Finger’, ‘Shine Muscat’, ‘Flame Seedless’, ‘Bixiang Wuhe’ and ‘Thompson Seedless’ showed sensitivity to high temperature. We further evaluated the heat tolerance of different grapevine cultivars by principal component analysis and the optimal segmentation clustering of ordered samples. These findings provide a theoretical basis for adopting appropriate cultivation management measures to reduce the effect of high temperatures and offer fundamental knowledge for future breeding strategies for heat-tolerant grapevine varieties.