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Temperatures worldwide have been rising due to global warming. The Korean grapevine (Vitis labruscana) cultivars are sensitive to high temperature during the vegetative growing period and are challenged with various physiological disorders, resulting in decreased production and poor quality of fruit. This study was conducted to identify genes that are differentially expressed in response to high temperature to help elucidate the mechanism of tolerance to temperatures in grapevines. 'Kyoho' grapevine leaves were maintained at 25 degrees C and 35 degrees C for 24 h in a plastic house and then used for transcriptome analysis by RNA-seq. More than 92 million transcripts were used for transcriptome analysis, with a total length of 7 billion bp and an average length of 89.6 bp; an average of 85.5% of the data were used in the final analysis. Investigation of differentially expressed genes (DEG) in response to high temperature discovered 243 induced genes and 378 inhibited genes. The selected DEGs were related to metabolism (49 genes), cell structure (22 genes), and molecular functions (15 genes). The selected DEGs clustered in the pathways of carbohydrate metabolism, amino acid metabolism, and the citrate cycle in the KEGG metabolic pathway. Among the selected DEGs, we selected the top 10 highly up-regulated and the top 10 down-regulated genes and confirmed their expression levels by reverse transcription quantitative PCR. At high temperature, the top 10 genes, including BCL-2-associated athanogene 6, heat shock protein 21, HAD superfamily, and subfamily IIIB acid phosphatase, showed induced expression, whereas the other 10 genes, including APS reductase3, ribosomal protein S7e family protein, and ribosomal protein L2 family, showed inhibited expression pattern. This is consistent with the results from the transcriptome analysis. We propose that these genes can potentially be used for the development of molecular markers to elucidate defense mechanisms against high temperature stress, and for breeding grapevine varieties tolerant to high temperature.