Heat stress causes disorders in plant growth and development, inhibits germination, and adversely affects photosynthetic pigments and lipid integrity of membranes. In response to stress, plants synthesize compatible osmolytes such as proline and heat shock proteins, and activate antioxidant mechanisms and epigenetic changes, including DNA methylation. The DMS3 protein is part of the RNA-directed DNA methylation system involved in de novo DNA methylation and is involved in the regulation of gene expression. The aim of this study was to investigate the effect of elevated temperatures (30, 32, 32/24 and 35 °C) on wild type plant Arabidopsis thaliana, the dms3-1 line with the mutated DMS3 gene and the oeDMS3 transgenic line (called 6TV) with overexpression of DMS3 gene, in order to determine if there are differences in their response to heat stress. Germination, radicle length and biomass were measured as morphological parameters, and content of photosynthetic pigments and proline, degree of lipid peroxidation (content of malondialdehyde) and presence of HSP70 protein as biochemical parameters. At elevated temperatures, the values of morphological parameters were significantly reduced in the dms3-1 mutant, compared to wild-type and 6TV line. The content of chlorophyll a and b, and carotenoids was significantly reduced at elevated temperatures in all lines. Higher values of malondialdehyde were observed in the dms3-1 mutant at all temperatures above 30 °C. The proline content did not differ significantly between the lines. The presence of HSP70 protein was more pronounced in the dms3-1 and 6TV lines at elevated temperatures. The results show that the dms3-1 line is more sensitive and the 6TV line is more resistant to heat stress, from which it can be concluded that DNA methylation mediated by RNA molecules plays a significant role in the adaptation of plants to heat stress.