Abstract The growth and tolerance of Kluyveromyces marxianus at high temperatures decreased significantly in the synthetic medium (SM), which is commonly used in industrial fermentations. After 100 days of adaptive laboratory evolution, a strain named KM234 exhibited excellent tolerance at a high temperature, without loss of its growth ability at a moderate temperature. Transcriptomic analysis revealed that the KM234 strain decreased the expression of the ammonium (NH4+) transporter gene MEP3 and increased the synthesis of the amino acid carbon backbone, which may contribute greatly to the high‐temperature growth phenotype. High NH4+ content in SM significantly increased the reactive oxygen species (ROS) production at high temperatures and thus caused toxicity to yeast cells. Replacing NH4+ with organic nitrogen sources or increasing the concentration of potassium ions (K+) in the medium restored the growth of the wild‐type K. marxianus at a high temperature in SM. We also showed that the NH4+ toxicity mitigated by K+ might closely depend on the KIN1 gene. Our results provide a practical solution to industrial fermentation under high‐temperature conditions.