Purpose: Restriction spectrum imaging (RSI-MRI), an advanced diffusion imaging technique, can potentially circumvent current limitations in tumor conspicuity, in vivo characterization, and location demonstrated by multiparametric magnetic resonance imaging (MP-MRI) techniques in prostate cancer detection. Prior reports show that the quantitative signal derived from RSI-MRI, the cellularity index, is associated with aggressive prostate cancer as measured by Gleason grade (GG). We evaluated the reliability of RSI-MRI to predict variance with GG at the voxel-level within clinically demarcated prostate cancer regions.Experimental Design: Ten cases were processed using whole mount sectioning after radical prostatectomy. Regions of tumor were identified by an uropathologist. Stained prostate sections were scanned at high resolution (75 mm/pixel). A grid of tiles corresponding to voxel dimensions was graded using the GG system. RSI-MRI cellularity index was calculated from presurgical prostate MR scans and presented as normalized z-score maps. In total, 2,795 tiles were analyzed and compared with RSI-MRI cellularity.Results: RSI-MRI cellularity index was found to distinguish between prostate cancer and benign tumor (t ¼ 25.48, P < 0.00001). Significant differences were also found between benign tissue and prostate cancer classified as low-grade (GG ¼ 3; t ¼ 11.56, P < 0.001) or high-grade (GG ! 4; t ¼ 24.03, P < 0.001). Furthermore, RSI-MRI differentiated between low and high-grade prostate cancer (t ¼ 3.23; P ¼ 0.003).Conclusions: Building on our previous findings of correlation between GG and the RSI-MRI among whole tumors, our current study reveals a similar correlation at voxel resolution within tumors. Because it can detect variations in tumor grade with voxel-level precision, RSI-MRI may become an option for planning targeted procedures where identifying the area with the most aggressive disease is important. Clin Cancer Res; 22(11); 2668-74. Ó2016 AACR.