Abstract Background: Global DNA methylation may affect chromosome structure and genomic stability and is involved in carcinogenesis. In this study, we aimed to investigate whether methylation of pericentromeric repeat (NBL2) and subtelomeric repeat (D4Z4) in peripheral blood leukocytes was associated with the aggressiveness of prostate cancer (PCa). Methods: We measured the methylation status of different CpG sites of NBL2 and D4Z4 in 900 PCa patients via pyrosequencing after bisulfite treatment. We then used multivariate logistic regression to estimate the odds ratio (ORs) and 95% confidence intervals (CIs) for analyzing the association of NBL2 and D4Z4 methylation with the aggressiveness of PCa at diagnosis. We then analyzed the association of CpG sites with the risk of biochemical recurrence (BCR) in patients receiving radical prostatectomy or radiotherapy using a multivariate Cox proportional hazards model adjusting for age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. In addition, we used the Kaplan-Meier survival function and log-rank tests to assess BCR-free survival associated with these markers. Results: There was no significant differences in the methylation level of NBL2 and D4Z4 between clinically defined aggressive and non-aggressive PCa at diagnosis. However, the methylation of subtelomeric region D4Z4 was associated with BCR, while the methylation of NBL2 was not associated with BCR. In tertile analysis, we found that patients with higher methylation of D4Z4 exhibited an increased risk of BCR in localized patients receiving definitive therapy. Because of the relatively low rate of BCR among patients, we combined the highest and second tertiles to increase statistical power. Patients in the second and third (highest) tertiles had an increased risk of BCR (HR=1.74, 95% CI, 1.14-2.65; p= 0.0096) compared to patients in the lowest tertile after adjustment of age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. We assayed 4 CpG sites in this region. In detailed analysis of each CpG site, the association was mostly attributable to the methylation of the 2nd methylation site of D4Z4 (HR=1.80, 95% CI, 1.19 - 2.72). Conclusion: These data suggest that methylation in the subtelomeric region D4Z4 may be able to predict worse prognosis of localized PCa patients. Citation Format: Yuyan Han, Jeri Kim, Xifeng Wu, Jian Gu. Methylation of subtelomeric repeat D4Z4 in peripheral blood leukocytes is associated with biochemical recurrence in localized prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2017-2396