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Abstract Background Robotic-assisted laparoscopic radical prostatectomy is a current standard treatment for localized prostate cancer, with treatment failure defined by biochemical recurrence (BCR). Open radical prostatectomy series have identified the presence of a positive surgical margin (PSM) as a predictor of long-term recurrence, a measure that is affected by the surgeon׳s skill. We evaluate the effect of PSM parameters on BCR rates from robotic-assisted laparoscopic radical prostatectomy, across 3 high-volume institutions. Methods De-identifiable clinicopathological and histopathological data were prospectively collected for 4,001 patients with at least 3 years of follow-up. Kaplan-Meier plots and 3 statistical models were used to evaluate the effect of margin parameters on BCR, via crude rates, traditional multivariable Cox regression, and a propensity-adjusted Cox regression model. Results Overall, 37% of men with a PSM developed BCR compared with 10% of men with negative margins (hazard ratio [HR] = 1.81, 95% CI: 1.47–2.22). Length ≥3 mm or a multifocal positive margin was associated with a higher risk of BCR compared with negative margin cases. On multivariable Cox regression analysis of the positive margin cohort, only apical margins significantly predicted BCR relative to basal margins (HR = 2.03, 95% CI: 1.01–4.09), whereas there was no significant difference in BCR rates for posterolateral margins relative to basal margins (HR = 1.62, 95% CI: 0.84–3.11). Propensity-adjusted modeling confirmed a greater effect of apical compared with posterolateral PSM. Conclusions A PSM length ≥3 mm is predictive of BCR, as is to a lesser extent multiple positive margins. In contrast to open prostatectomy series, posterolateral margins carry a smaller risk of BCR compared with apical margins.