PURPOSE: Approximately 35% of patients with prostate cancer who undergo radical prostatectomy experience prostate specific antigen recurrence within 10 years of surgery. Current prognostic indicators cannot sufficiently detect who is at risk for biochemical recurrence. We evaluated DNA methylation markers for prostate cancer prognosis. MATERIALS AND METHODS: We assessed the DNA methylation of 6 marker candidates that were identified in previous studies. Formalin fixed, paraffin embedded tissue sections from a cohort of 605 patients who underwent radical prostatectomy were analyzed using real-time polymerase chain reaction assays. Using a Cox proportional hazard model we determined which markers were significant predictors of biochemical recurrence. RESULTS: ABHD9, Chr3-EST, GPR7, HIST2H2BF and PITX2 were significantly associated with biochemical recurrence. PITX2 methylation was the strongest predictor of biochemical recurrence, providing additional prognostic information to established clinical factors in patients treated with radical prostatectomy and especially in patients at intermediate risk (Gleason 7). Patients with greater than median PITX2 methylation in the tumors were 4 times more likely to experience biochemical recurrence within 8 years after surgery than patients with less than average methylation. CONCLUSIONS: The prognostic information provided by PITX2 methylation adds significantly to currently used clinical variables such as Gleason grade and stage. Therefore, it could contribute to better counseling in patients with prostate cancer.