Forty-four yeast isolates were tested for thermo-tolerance at 30, 36, 39, 42, 43, 44 and 45ºC and for ethanol tolerance at 4, 8, 10 and 12% (v/v) of ethanol. Seven yeast isolates (C2, CC, BM2, V2, V3, L04-2 and L07-2) were found to be able for growth at 43ºC, of which BM2 could grow at 44ºC. Twenty-five yeasts could grow in the medium containing 12% (v/v) ethanol. Among them, V2 performed the superior ethanol fermentation. Favorable conditions for V2 in ethanol production from molasses at 40ºC were determined as follow: 105 cells/mL of inoculum level, 25ºBrix of initial sugar concentration, 5 days of fermentation and at pH 4.66. The ethanol concentration obtained could reach at 4.48% (w/v) in fermentation from molasses medium at 40ºC. The result of molecular analysis of ITS1, 5.8S rDNA, and ITS2 sequence showed that the V2 yeast isolate belonged to Pichia kudriavzevii with 100% homogeneous level.