Membrane fatty acid composition has an important role in yeast stress resistance, particularly in temperature tolerance. Most studies investigating temperature and membrane fatty acids use the yeast Saccharomyces cerevisiae without considering other yeasts, such as Kluyveromyces marxianus, which has physiological differences and industrial advantages with respect to S. cerevisiae. One of the primary traits of K. marxianus is its thermotolerance. The effect of fatty acid addition (oleic acid, linoleic acid, linolenic acid and araquidic acid) on the thermotolerance of the K. marxianus strain SLP1 was evaluated. SLP1 yeast exhibited temperature tolerance of up to 50°C; at 55°C, viability was reduced significantly, probably due to an increase in the generation of reactive oxygen chemical species. Externally added fatty acids were incorporated in the yeast membrane, increasing their proportion to approximately 70%, thereby changing membrane fluidity. SLP1 cells supplemented with polyunsaturated fatty acids decreased cell thermotolerance and increased the degree of lipoperoxidation, while arachidic acid addition exhibited a tendency to increase yeast thermotolerance.