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To investigate diffusion anisotropy in whole human prostate specimensSeven whole radical prostatectomy specimens were obtained with informed patient consent and institutional ethics approval. Diffusion tensor imaging was performed at 9.4 Tesla. Diffusion tensors were calculated from the native acquired data and after progressive downsamplingFractional anisotropy (FA) decreased as voxel volume increased, and differed widely between prostates. Fixation decreased mean FA by ˜0.05–0.08 at all voxel volumes but did not alter principle eigenvector orientation. In unfixed tissue high FA (> 0.6) was found only in voxels of volume <0.5 mm3, and then only in a small fraction of all voxels. At typical clinical voxel volumes (4–16 mm3) less than 50% of voxels had FA > 0.25. FA decreased at longer diffusion times (Δ = 60 or 80 ms compared with 20 ms), but only by ˜0.02 at typical clinical voxel volume. Peripheral zone FA was significantly lower than transition zone FA in five of the seven prostatesFA varies widely between prostates. The very small proportion of clinical size voxels with high FA suggests that in clinical DWI studies ADC based on three-direction measurements will be minimally affected by anisotropy. Magn Reson Med 76:626–634, 2016. © 2015 Wiley Periodicals, Inc.