Background: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance. Purpose: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy. Methods: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward. Results: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 32400000-32599999 were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 67800000-67999999 (P = .013), chromosome 3 140200000-140399999 (P = .037), and chromosome 12 101200000-101399999 (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 32400000-32599999 could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 149600000-149799999 were 0.066 and 0.076, respectively. Conclusion: Methylation change in chromosome 6 149600000-149799999 is an ideal predictor of exemestane resistance with great clinical potential.