Abstract Background Exposure to cytotoxic chemotherapy treatment may alter DNA methylation (DNAm) in treated patients. Methods We performed DNAm analysis in 1,244 and 897 breast cancer patients treated and not treated by adjuvant chemotherapy using the Illumina MethylationEPIC array (1,804 blood, 337 saliva). DNAm changes of 620,095 individual CpGs and 41,581 promoters were evaluated using linear regression models, adjusting for age at diagnosis, ethnicity, years between sample collection and diagnosis and cell-type heterogeneity. Results from datasets normalized separately were combined by meta-analysis (random effects model). Gene set enrichment analyses were conducted to identify key processes or pathways associated with chemotherapy treatment. Results A total of 425 differentially methylated CpGs and 20 promoters were significantly associated with chemotherapy treatment (p< 5e-8). Enriched gene sets among 3,495 chemotherapy-associated promoters (unadjusted p< 0.05, preranked by Z scores) included three suppressed Gene Ontology (GO) terms that survived Bonferroni correction (GO:0002376, immune system process; GO:0009605, response to external stimulus; and GO:1903034: regulation of response to wounding). Using meta-analysis regression coefficients for all promoters as a ranking metric, olfactory transduction (KEGG, hsa04740) was found to be significantly suppressed (unadjusted p=6.38e-06, adjusted p=0.002). Taste transduction (hsa04742, unadjusted p=1.73e-03, adjusted p=0.565) was the next most significantly suppressed pathway. Conclusion The enrichment of imprinted genes within biological processes and pathways suggests a biological mechanism by which chemotherapy treatment could affect immune response, wound healing and changes in the perceptions of smell and taste. Citation Format: Peh Joo Ho, Alexis Jiaying Khng, Benita Kiat Tee Tan, Ern Yu Tan, Geok hoon Lim, Su-Ming Tan, Veronique Kiak Mien Tan, Elaine Hsuen Lim, Mikael Hartman, Jingmei Li. Alteration of DNA methylation landscape in breast patients treated with adjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-44.