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Thermotolerant ethanologenic yeasts receive attention as alternative bio-ethanol producers to traditionally used yeast, Saccharomyces cerevisiae. Their utilization is expected to provide several benefits for bio-ethanol production due to their characteristics and robustness. They have been isolated from a wide variety of environments in a number of ASEAN countries: Thailand, Vietnam, Laos, and Indonesia. One of these yeasts, Kluyveromyces marxianus has been investigated regarding characteristics. Some strains efficiently utilize xylose, which is a main component of the 2nd generation biomass. In addition, the genetic basis of K. marxianus has been revealed by genomic sequencing and is exploited for further improvement of the strains by thermal adaptation or gene engineering techniques. Moreover, the glucose repression of K. marxianus and its mechanisms has been investigated. Results suggest that K. marxianus is an alternative to S. cerevisiae in next-generation bio-ethanol production industry. Indeed, we have succeeded to apply K. marxianus for bio-ethanol production in a newly developed process, which combines high-temperature fermentation with simultaneous fermentation and distillation under low pressure. This chapter aims to provide valuable information on thermotolerant ethanologenic yeasts and their application, which may direct the economic bioproduction of ethanol and other useful materials in the future.Recently, thermotolerant microorganisms were found among mesophiles with optimum growth temperatures that are 5-10°C higher than those of the typical mesophilic strains Fuel Ethanol Production from Sugarcane 122 Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries… http://dx.doi.org/10.5772/intechopen.79144 123Recently, there have been several reports on ethanol production at high temperatures using P. kudriavzevii (formerly known as I. orientalis). Several P. kudriavzevii strains were reported to grow and produce high levels of ethanol at high temperatures. The strain DMKU 3-ET15 was isolated from traditional fermented pork sausage in Thailand by an enrichment technique in a medium supplemented with 4% ethanol at 40°C. The strain produced 78.6 g/L ethanol from 180 g/L glucose at 40°C [20]. The strain KVMP10 that was isolated from soil located beneath apple trees for ethanol production from orange peel achieved 54 g/L ethanol at 42°C [48]. Strain RZ8-1 that was recently isolated from various samples collected from plant orchards in Thailand produced 33.8 g/L ethanol from 160 g/L glucose at 40°C [49].