The evaluation of plant stress tolerance and the screening of key regulatory genes under the combined stresses of high temperature and drought are important for the study of plant stress tolerance mechanisms. In this study, the drought tolerance of five grape varieties was evaluated under high-temperature conditions to screen key genes for further exploration of resistance mechanisms. By comparing and analysing the morphological characteristics and physiological indicators associated with the response of grapevines to drought stress and integrating them with the membership function to assess the strength of their drought tolerance, the order of drought tolerance was found to be as follows: 420A>110R>CS>fercal>188-08. To further analyse the mechanism of differences in drought tolerance, transcriptomic sequencing was performed on the drought-tolerant cultivar 420A, the drought-sensitive cultivar 188-08 and the control cultivar CS. The functional analysis of differential metabolic pathways indicated that the differentially expressed genes were enriched mainly in biological that 420A had higher antioxidant activity. Moreover, the transcription factors which differentially expressed were also analysed in the five grape varieties, and several genes, such as VvAGL15, VvLBD41, and VvMYB86, appeared to be closely related to drought tolerance, suggesting their potential involvement in the regulation of grapevine drought tolerance and their value in drought tolerance research.