At best of our knowledge, no previous study compared MLH1, MGMT, CDKN2A, and RASSF1A gene methylation levels among thymoma samples, healthy thymic samples, and circulating blood DNA from the same patients, and the present study revealed that MLH1, MGMT, CDKN2A, and RASSF1A genes showed similar and very low methylation patterns in the three investigated tissues, and no difference was observed between pathological and healthy thymus. This evidence concerns the gene MGMT and thymoma.