DNA methylation is responsive to various biotic and abiotic stresses. Heat stress is a serious threat to crop growth and development worldwide. Heat stress results in an array of morphological, physiological and biochemical changes in plants. The relationship between DNA methylation and heat stress in crops is relatively unknown. We investigated the differences in methylation levels and changes in the cytosine methylation patterns in seedlings of two rapeseed genotypes (heat-sensitive and heat-tolerant) under heat stress. Our results revealed that the methylation levels were different between a heat-tolerant genotype and a heat-sensitive one under control conditions. Under heat treatment, methylation increased more in the heat-sensitive genotype than in the heat-tolerant genotype. More DNA demethylation events occurred in the heat-tolerant genotype, while more DNA methylation occurred in the heat-sensitive genotype. A large and diverse set of genes were affected by heat stress via cytosine methylation changes, suggesting that these genes likely play important roles in the response and adaption to heat stress in Brassica napus L. This study indicated that the changes in DNA methylation differed between heat-tolerant and heat-sensitive genotypes of B. napus in response to heat stress, which further illuminates the molecular mechanisms of the adaption to heat stress in B. napus.