Little is known about the mechanisms causing loss of virulence in pathogenic fungi as a result of protracted culture. We studied the extent to which patterns of DNA methylation varied between virulent and reduced virulence derivative cultures of Botrytis cinerea, and identify the genes/genomic regions affected by these epigenetic modifications. B. cinerea was cultured in vitro for eight months involving subculture every four weeks. Fungal conidia were harvested at every four-week subculturing stage and inoculated onto Arabidopsis thaliana Col-0 plants for virulence testing. Global epi/genetic changes in B. cinerea during culture were assessed using methylation-sensitive amplified polymorphisms (MSAPs) on mycelium from eight different sub-culture time points and from mycelium recovered after eight months in culture and then inoculated onto A. thaliana. Culture induced epi/allele characterisation was carried out by whole genome sequencing and bisulfite sequencing of gDNA from samples after two and eight months in culture and after 8 months in culture and following inoculation onto an A. thaliana plant. Virulence declined with time in culture and recovered after one fungal generation on A. thaliana. MSAP data 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.