Most of the yeasts are mesophilic organisms with optimal growth temperatures around 28–30 °C. There are few yeast species which tolerate much higher growth temperatures, with Ogataea polymorpha and Kluyveromyces marxianus being the best known and characterized. Both mentioned species grow maximally at near 50 °C and are used commercially for production of biofuels and recombinant proteins. They offer several important advantages relative to mesophilic yeasts, e.g. could be suitable for simultaneous saccharification and fermentation (SSF) of lignocellulosic residues or other polymers. In spite of biotechnological importance, mechanisms of thermotolerance of O. polymorpha and K. marxianus are poorly understood. It was found that activation of trehalose synthesis and overexpression of heat shock proteins improve growth at sublethal temperature (50 °C) of O. polymorpha still having no effect on maximal growth temperature. In K. marxianus (but not in O. polymorpha), shift from 30 °C to 45 °C changes glucose metabolic flow from glycolysis to pentose phosphate pathway. Mostly, mechanisms of yeast thermotolerance are studied on mesophilic yeasts like Saccharomyces cerevisiae and some other species. For this, adaptive laboratory evolution (ALE) for more thermotolerant strains with next whole-genome sequencing is used. Besides, transformation of S. cerevisiae with gene library of O. polymorpha and selection for thermotolerant transformants have been applied. These and other approaches showed that thermotolerance in yeast is a complex phenotype dependent on multiple quantitative loci. It was found that yeast heat resistance depends on at least six genomic changes, including gene expression responses, heat shock proteins, trehalose, ATPase, the ubiquitin-proteasome pathway, and heat-induced antioxidant defences. Thermotolerance was shown to be elevated due to deletion of genes SIN3, SRB2, and MIG1 coding for transcription factors, ERG5, ERG4, and ERG3 involved in ergosterol synthesis, by expression of PDR18 gene coding for ABC transporter, overexpression of RSP5 gene coding for ubiquitin ligase, due to mutations in cell cycle CDC25 gene, the SWI/SNF complex, and F-type ATPase or characterized by higher sphingolipid content or activated one-carbon metabolism relative to the wild-type strains. Decrease ploidy from 4N to 1N also elevated thermotolerance. The list of genes and mutations leading to improved thermotolerance could be continued; however, exact mechanisms in each case, as a rule, remain elusive. Interactions (if any) between identified genes/products also remain to be studied. Mechanisms of yeast thermotolerance have important basic interest and practical value due to many biotechnological reasons and are actively investigated.