The thermotolerant yeast Kluyveromyces marxianus is a promising bioethanol producer, but its fermentation arrested earlier at high temperatures. Untargeted metabolomic analysis was used to explore K. marxianus's stress responses during high-temperature fermentation. Fermentation experiments were conducted at 45 degrees C, with a group conducted at 30 degrees C as control. Samples were collected from both groups at 14 and 22 h and were subjected to metabolomic analysis. The results show that pathways related to biosynthesis of amino acids, metabolism of purine, pyridoxine and riboflavin, etc. were induced at high temperature. More oleic acid, less palmitic acid and less palmitoleic acid were synthesized at 45 degrees C than at 30 degrees C, indicating that K. marxianus can adapt to high temperature by increasing the mean fatty acid chain length. In addition, most of the metabolic pathways stopped after the fermentation arrest, but pathways related to amino acids metabolism, riboflavin metabolism, etc. were still active. This is the first study about comprehensive metabolomic profiling of K. marxianus during high-temperature fermentation. The findings in this study offer deeper insight in mechanisms of K. marxianus's stress responses during high-temperature fermentation at the metabolome level, and provide potential target pathways for further metabolic engineering towards improved stress tolerance and efficient bioethanol production.