Abstract Purpose: Fusion of the TMPRSS2 gene with the ERG oncogene and aberrant DNA methylation patterns are commonly found in prostate cancer. The aim of this study was to analyze the relationship between ERG expression, DNA methylation of three biomarkers, and clinicopathologic features of prostate cancer. Experimental Design: Immunohistochemistry for ERG protein was conducted as a surrogate for TMPRSS2-ERG fusions. We analyzed methylation of CYP26A1, TBX15, and HOXD3 in 219 prostatectomy specimens by the quantitative MethyLight assay. DNA methylation was compared between ERG-positive and -negative cases and correlations of ERG and DNA methylation with clinicopathologic features were analyzed using χ2, Spearman correlation, logistic regression, and Cox regression. Results: ERG expression varied according to Gleason pattern (almost absent in pattern II, highest in pattern III, and lower in pattern IV/V) and showed a strong positive correlation with methylation levels of CYP26A1, TBX15, and HOXD3 (Spearman P < 0.005). TBX15 and HOXD3 methylation were significantly associated with pathologic stage, Gleason score, and Gleason pattern (P ≤ 0.015). In multivariate regression analysis, PSA, TBX15 high methylation, and HOXD3 high methylation were significantly associated with stage (P < 0.05), whereas ERG expression was negatively correlated with Gleason score (P = 0.003). In univariate time-to-recurrence analysis, a combination of HOXD3/TBX15 high methylation predicted recurrence in ERG-positive and -negative cases (P < 0.05). Conclusions: CYP26A1, TBX15, and HOXD3 are methylation markers of prostate cancer associated with ERG expression and clinicopathologic variables, suggesting that incorporation of these markers may be useful in a pre- and posttreatment clinical setting. Clin Cancer Res; 18(10); 2896–904. ©2012 AACR.