Moderate heat stress may provide protection against a subsequent severe high temperature stress in plants. However, the exact mechanisms of heat acclimation of wheat are still poorly understood. In the present work, two wheat varieties Ellvis and Soissons were exposed to a moderate elevated temperature at 30 °C, and the changes of certain protective mechanisms were investigated. Although the differences in the proline level between the genotypes were not substantial, it was approx. 2-3 times higher in the heat-treated plants than in the controls. After exposure to moderate elevated temperature, the activities of ascorbate peroxidase and catalase were also induced. Similarly, the amount of the free salicylic acid also increased after moderate heat stress, independently on the genotypes. The amount of the main polyamines, namely, putrescine, spermidine, and spermine either did not change or decreased after the same period. However, heat acclimation increased the level of 1,3-diaminopropane, in parallel with a polyamine oxidase gene, TaPAO. While the expression level of the peroxisomal polyamine oxidase gene TaperPAO hardly changed, TaPAO showed a substantial increase after 1 day, especially in Soissons, and at the end of the heat treatment was still significantly higher than in the controls. These suggest that signalling processes related to polyamine metabolisms or salicylic acid-related processes might also contribute to the higher heat tolerance induced by moderate heat stress. The variations in recorded measurements were mainly temperature dependent, and the effect of genotype was less pronounced than the effect of moderate heat treatment itself.