Abstract Introduction: The detection of methylated tumor suppressor genes (TSGs) in ductal aspirates/lavages from breast cancer (BC) patients suggests a role for DNA methylation in molecular screening. We measured DNA methylation in normal tissue from non-cancer patients (Nl), normal tissue from BC patients (Nl-CA), and malignant tissue from patients with in situ (DCIS) or invasive (CA) BC to determine whether methylation changes can distinguish between normal and cancer. Methods: DNA was extracted and bisulfite converted from fresh frozen tissue (reduction mammoplasty (Nl, 7), matched Nl-CA and CA (48)) or microdissected paraffinized tissue (Nl from benign biopsy (3), or DCIS (7)). Methylation of APC, ESR1, HPP1, and p16 was assayed using the MethyLight assay. Results: All Nl specimens were unmethylated. Methylation of the 4 TSGs was observed in tissues from cancer patients (Table). The mean number of methylated genes (NMG) increased from Nl (0) to Nl-CA (0.5), to cancer (DCIS 1.1, CA 1.2) (p Conclusions: The increased NMG from Nl to cancer suggests a progressive accumulation of methylation with malignant progression. Nl-CA tissue displays elevated levels of TSG methylation, suggesting a methylation field effect. When assessing several genes simultaneously, methylation can distinguish between normal and cancer tissue. The discriminatory power will likely increase with the number of genes assayed. These findings support the evaluation of DNA methylation as a biomarker for molecular screening.