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New industrial microorganisms are required to improve biofuel production with a key factor being ethanol fermentation at a high temperature. In this study, 222 yeast isolates from sugarcane field soil from lower Northern Thailand were isolated using yeast extract peptone dextrose medium supplemented with 4% (v/v) ethanol. The isolates were characterized for growth performance under stress conditions and for fermentation activity at high temperatures. A phylogenetic analysis of 25 thermotolerant yeasts grouped the isolates into clusters of Candida blankii, Saccharomyces cerevisiae and Pichia kudriavzevii. Two isolates, P. kudriavzevii NUPHS33 and NUPHS34, exhibited high temperature and ethanol tolerance levels of up to 45 °C and 15 (v/v), respectively. Ethanol production upon utilization of glucose as a carbon source was determined by gas chromatography. The highest ethanol concentration of P. kudriavzevii NUPHS33 and NUPHS34 reached 61.51-1.01 g/l at 45 °C and 69.79-1.54 g/l at 40 °C, respectively, which is significantly greater than the amount of ethanol produced by the industrial strain S. cerevisiae TISTR5606 (p < 0.01). Therefore, the newly isolated thermo- and ethanol-tolerant P. kudriavzevii NUPHS33 and NUPHS34 are highly competent yeasts for high-temperature ethanol fermentation. Our results might be useful to further improve commercial-scale bioethanol production. [ABSTRACT FROM AUTHOR]