© 2015 Liss, White, Parsons, Schenker-Ahmed, Rakow-Penner, Kuperman, Bartsch, Choi, Mattrey, Bradley, Shabaik, Huang, Margolis, Raman, Marks, Kane, Reiter, Dale and Karow. Purpose: We evaluate a novel magnetic resonance imaging (MRI) technique to improve detection of aggressive prostate cancer (PCa). Materials and Methods: We performed a retrospective analysis of pre-surgical prostate MRI scans using an advanced diffusion-weighted imaging technique called restriction spectrum imaging (RSI), which can be presented as a normalized z-score statistic. Scans were acquired prior to radical prostatectomy. Prostatectomy specimens were processed using whole-mount sectioning and regions of interest (ROIs) were drawn around individual PCa tumors. Corresponding ROIs were drawn on the MRI imaging and paired with ROIs in regions with no pathology. RSI z-score and conventional apparent diffusion coefficient (ADC) values were recorded for each ROI. Paired t-test, ANOVA, and logistic regression analyses were performed. Results: We evaluated 28 patients with 64 ROIs (28 benign and 36 PCa). The mean difference in RSI z-score (PCa ROI-Benign ROI) was 2.17 (SE = 0.11; p < 0.001) and in ADC was 551 mm2/s (SE = 80 mm2/s; paired t-test, p < 0.001). The differences in the means among all groups (benign, primary Gleason 3, and primary Gleason 4) was significant for both RSI z-score (F3,64= 97.7, p < 0.001) and ADC (F3,64= 13.9, p < 0.001). A t-test was performed on only PCa tumor ROIs (n = 36) to determine PCa aggressiveness (Gleason 3 vs. Gleason 4) revealing that RSI z-score was still significant (p = 0.03), whereas, ADC values were no longer significant (p = 0.08). In multivariable analysis adjusting for age and race, RSI z-score was associated with PCa aggressiveness (OR 10.3, 95% CI: 1.4-78.0, p = 0.02) while ADC trended to significance (p = 0.07). Conclusion: The RSI-derived normalized cellularity index is associated with aggressive PCa as determined by pathologic Gleason scores. Further utilization of RSI techniques may serve to enhance standardized reporting systems for PCa in the future.