Thermotolerant ethanologenic yeasts have attracted the interest of many scientists due to the current challenges caused by increasing global temperature, the benefits associated with processing at high temperatures, and the potential to reduce cooling costs. The objectives of this study are to characterize the selected thermotolerant yeasts and to evaluate their use in Cayratia trifolia fermentation at high temperatures. A total of 151 yeast strains isolated from 53 samples of Cayratia trifolia were studied for their morphology, physiology, biochemistry, and their phylogenetic relationship. Based on the results of tests for thermotolerance ability (37-45oC) and ethanol tolerance capacity (9-12% v/v), 57 of the 151 yeast isolates were selected to be tested for use in wine fermentation from three-leaf cayratia at 37oC. Thirty isolates that were found to have high fermentation ability and that produced an ethanol concentration of between 6.0 and 9.9% (v/v) were selected for identification using amplified 26S rDNA sequences. The yeasts were identified as follows: Candida glabrata (BL2.1, CT1.1, CT1.3, CT2.3, HG2.1), Candida tropicalis (KG1.1, KG3.2, CM3.3, HG3.3, TG1.1, TG3.1), Candida nivariensis (DT1.2, CM3.2, ST2.1, BT1.2), Pichia kudriavzevii (KG2.1, KG5.1, AG2.1, AG2.3, AG4.2, DT3.2, LA1.3, CM4.4, BT2.1, BT3.3, TV4.2, CT4.2, VL1.1), Clavispora lusitaniae (TG4.2), and Saccharomyces cerevisiae (HG1.3). The phylogenetic tree constructed using MEGA 6 with bootstrap analysis performed by repeating the data 1,000 times revealed that the selected yeast strains were closely related. The newly isolated strain of S. cerevisiae HG1.3 producing the highest ethanol concentration of 9.9% (v/v) in Cayratia trifolia wine fermentation at 37oC was selected for further study.