Background Tissue estimates obtained by using microstructure imaging techniques, such as hybrid multidimensional (HM) MRI, may improve prostate cancer diagnosis but require histologic validation. Purpose To validate prostate tissue composition measured by using HM MRI, with quantitative histologic evaluation from whole-mount prostatectomy as the reference standard. Materials and Methods In this HIPAA-compliant study, from December 2016 to July 2018, prospective participants with biopsy-confirmed prostate cancer underwent 3-T MRI before radical prostatectomy. Axial HM MRI was performed with all combinations of echo times (57, 70, 150, and 200 msec) and b values (0, 150, 750, and 1500 sec/mm2). Data were fitted by using a three-compartment signal model to generate volumes for each tissue component (stroma, epithelium, lumen). Quantitative histologic evaluation was performed to calculate volume fractions for each tissue component for regions of interest corresponding to MRI. Tissue composition measured by using HM MRI and quantitative histologic evaluation were compared (paired t test) and correlated (Pearson correlation coefficient), and agreement (concordance correlation) was assessed. Receiver operating characteristic curve analysis for cancer diagnosis was performed. Results Twenty-five participants (mean age, 60 years ± 7 [standard deviation]; 30 cancers and 45 benign regions of interest) were included. Prostate tissue composition measured with HM MRI and quantitative histologic evaluation did not differ (stroma, 45% ± 11 vs 44% ± 11 [P = .23]; epithelium, 31% ± 15 vs 34% ± 15 [P = .08]; and lumen, 24% ± 13 vs 22% ± 11 [P = .80]). Between HM MRI and histologic evaluation, there was excellent correlation (Pearson r: overall, 0.91; stroma, 0.82; epithelium, 0.93; lumen, 0.90 [all P < .05]) and agreement (concordance correlation coefficient: overall, 0.91; stroma, 0.81; epithelium, 0.90; and lumen, 0.87). High areas under the receiver operating characteristic curve obtained with HM MRI (0.96 for epithelium and 0.94 for lumen, P < .001) and histologic evaluation (0.94 for epithelium and 0.88 for lumen, P < .001) were found for differentiation between benign tissue and prostate cancer. Conclusion Tissue composition measured by using hybrid multidimensional MRI had excellent correlation with quantitative histologic evaluation as the reference standard. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Muglia in this issue.