Purpose: The goal of this study is to validate the muscle architecture derived from both ex vivo and in vivo diffusion-weighted magnetic resonance imaging (dMRI) of the human tongue with histology of an ex vivo tongue. Method: dMRI was acquired with a 200-direction high angular resolution diffusion imaging (HARDI) diffusion scheme for both a postmortem head (imaged within 48 hr after death) and a healthy volunteer. After MRI, the postmortem head was fixed and the tongue excised for hematoxylin and eosin (H&E) staining and histology imaging. Structure tensor images were generated from the stained images to better demonstrate muscle fiber orientations. The tongue muscle fiber orientations, estimated from dMRI, were visualized using the tractogram, a novel representation of crossing fiber orientations, and compared against the histology images of the ex vivo tongue. Results: Muscle fibers identified in the tractograms showed good correspondence with those appearing in the histology images. We further demonstrated tongue muscle architecture in in vivo tractograms for the entire tongue. Conclusion: The study demonstrates that dMRI can accurately reveal the complex muscle architecture of the human tongue and may potentially benefit planning and evaluation of oral surgery and research on speech and swallowing.