Canola (Brassica napus) is an important crop species grown worldwide for its seeds, which are crushed for edible oil. Canola production is threatened by blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, which can lead up to 100% yield loss. A plant’s disease resistance response depends highly on the regulation of the expression of defence genes. DNA methylation, an epigenetic mark, is one of the most important regulatory mechanisms in a plant’s defence system. Here, the DNA methylation pattern of promoters of defence genes has been investigated between leaves from control and infected plants with L. maculans of resistant and susceptible cultivars of B. napus. In total, over 12,000 promoters were found to be differentially methylated between leaves from control and infected plants in the two cultivars, of which 225 promoters were related to defence genes and differentially methylated between the resistant and susceptible cultivars. The majority of defence gene promoters were hypo- or hyper-methylated in the first and second true leaves, but not in the third and fourth leaves. The outcomes will assist in developing an insight into genome-wide DNA methylation patterns in canola cultivars, and ultimately help breeders to optimise the breeding programmes for enhancing resistance against blackleg disease.