PURPOSE: Digital image analysis software to review histopathology specimens is advancing uropathology by objectivity and reproducibility. Subjective pathologist assessed prostate tumor volume calculations correlate with known prognostic factors at radical prostatectomy. We ascertained whether image analysis software calculations of prostate tumor volume correlate with such prognostic factors, particularly positive surgical margins. MATERIALS AND METHODS: Prostate tumor volume was calculated using digital image analysis software in 851 sequential radical prostatectomy specimens. Results were correlated with clinicopathological data by logistic regression. RESULTS: Median prostate tumor volume was 2.2 cc (IQR 0.9-3.8). Median peripheral and transition zone tumor volume was 1.9 and 0.6 cc, respectively. Transition zone tumors were noted in 236 specimens (27.8%) and positive surgical margins occurred in 244 (28.7%). Tumors with extensive positive surgical margins had larger image analysis software assessed prostate tumor volume (p = 0.029) and peripheral zone volume (p = 0.007) than those with only focal positive surgical margins. On univariate analysis positive surgical margin tumors were larger and had seminal vesicle invasion, extraprostatic extension, perineural invasion and higher preoperative prostate specific antigen than those without positive surgical margins (each p <0.001). A linear relationship existed between image analysis software determined prostate tumor volume, and increasing tumor stage, Gleason score and prostate specific antigen (p for trend <0.001). On multivariate analysis tumor volume and tumor volume as a percent of prostate volume predicted positive surgical margins. CONCLUSIONS: Prostate tumor volume determined objectively by image analysis software correlates with positive surgical margins, as do prognostic variables such as extraprostatic extension, seminal vesicle invasion, perineural invasion, peripheral zone volume and Gleason score greater than 7. Objective digital image analysis software assessment appears to be a valid form to determine prostate tumor volume at radical prostatectomy. It is a useful adjunct to other histopathological analyses.