Abstract DNA methylation is an essential epigenetic mechanism involved in gene regulation. Aberrant DNA methylation plays an important role in breast cancer initiation and progression via inactivation of tumor suppressor genes and activation of oncogenes. Conventional approaches comparing DNA methylation profiles in tumor and adjacent non-tumorous tissue have shown that changes in DNA methylation contribute to deregulation of gene expression in breast cancer. However, adjacent non-tumorous tissue represents a suboptimal baseline control because its molecular profile may have been altered, and to some extent, similar to that of tumor. Consequently, important breast cancer-associated DNA methylation may not be identifiable in such comparisons. Using RNA-sequencing data generated from freshly-frozen breast tissue samples, we examined expression of methylation-related genes in 119 breast tumor, 47 adjacent normal-appearing breast tissue samples, and 23 normal breast tissue samples from healthy donors. Bowtie and RESM algorithms were used to align raw reads and quantify transcript abundance. Differential expression analyses were performed using edgeR software and normalized transcript counts. Normal breast tissue from healthy women compared to tumor from cancer patients identified differentially expressed cancer genes that are methylated in tumor, such as BRCA1, ESR1, ESR2, CDKN2A, HIC1, CDCP1, and SFRP1. A subset of these genes were found to be deregulated in adjacent non-tumorous tissue in a similar way as in tumor tissue, including PTGS2, SFN, SYK, FHIT , CST6, GADD45A, and THBS1, and are thus undetectable in the comparison between tumor and adjacent non-tumor tissue. In addition, a set of methylation-related genes were identified only in the comparison between tumor and normal tissue, including genes directly involved in the methylation/epigenetic process (HIST1H4A, KDM1B, MAT2B and SAP18) as well as novel target genes that may be methylated and play an important role in carcinogenesis (NFIA, ARHGAP18, and GRHL2). These findings suggest that normal breast tissue from healthy women is the most desirable control in comparison with tumor tissue to identify novel, tissue-specific methylation changes that may be important for carcinogenesis. Citation Format: Erin Wagner, Wenting Wu, Yunlong Liu, Kenneth Nephew, Jiali Han, Chunyan He. DNA methylation in tumor, adjacent non-tumorous and normal breast tissues. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A51.