Utilization of thermotolerant microorganisms enables fermen- tation to be performed at high temperatures, which have several advantages such as reduce cost for cooling system, reduce contam- ination of mesophilic microorganisms and increase the speed of catalytic reactions related to fermentation. In this work, isolation andcharacterizationofthermotolerantyeastscapableofproducing ethanol from Jerusalem artichoke (Helianthus tuberosus L.), one of the most suitable materials for bioethanol production as it con- tains nearly 20% of carbohydrates, were investigated. Soils and plantmaterialscollectedfromJerusalemartichokeplantationwere subjected to isolation of the thermotolerant yeast using enrich- ment culture technique. As the results, fifty isolates of yeast were obtained and they were maintained on YM agar. Among these iso- lates,onlysixisolateswereabletouseinulin,amajorcarbohydrate compound contained in Jerusalem artichoke tuber, as carbon and energy source for growth. The tolerance ability of these six isolated yeasts to high ethanol concentration and high temperature were tested by culturing them in YM medium containing various con- centrations of ethanol (0–10%, v/v) or by incubating at different temperatures (30–50◦C). As the results, all six isolated yeasts were able to grow in medium containing up to 4% ethanol at initial con- centration and up to 46oC of incubation temperature. Their ability to grow at high temperature indicating that these isolated yeasts are thermotolerant yeasts. Identification of the yeast strains was carried out using morphological and D1/D2 domain of 26S rDNA sequencing analysis and the results revealed that these isolated yeastswereKluyveromycesmarxianus.Ethanolfermentationability of the six isolated yeasts was compared using Jerusalem artichoke juice without acid or enzymatic pre-treatment as a raw material. The results showed that all isolates were produced relatively high ethanol concentration after 4 days of fermentation, however the highest ethanol concentration (8.0% v/v) was obtained from strain A102 and A103. These results suggested that the newly thermotol- erantyeasts,K.marxianusstrainA102andA103,havehighpotential for ethanol production from Jerusalem artichoke at high tempera- ture.