Pathogenic fungi can lose virulence after protracted periods of culture but little is known of the mechanisms regulating this. Here we assess whether DNA methylation could play a role in this phenomenon by the methylome analysis of virulent and reduced virulence derivative cultures of Botrytis cinerea, and identify the genes/genomic regions affected by these epigenetic modifications. Virulence declined during the eight months culture and recovered after one fungal generation on A. thaliana. Methylation-sensitive amplified polymorphisms show that epi/genetic variation followed virulence changes during culture. Whole genome sequencing showed no significant genetic changes during culture. Conversely, bisulfite sequencing showed significant changes both on global and local methylation patterns. We suggest that virulence is a non-essential plastic character regulated by DNA methylation during protracted in vitro culture. We propose DNA methylation as a regulator of the high virulence/low virulence transition in B. cinerea and as a potential mechanism to control pathogenicity.