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To study the anatomical relationships involving the intrinsic and extrinsic myofiber populations of the human tongue employing diffusion tensor imaging (DTI) with tractography.Images of the human tongue in vivo were obtained using a twice-refocused spin echo DTI pulse sequence at 1.5T, isotropic 3 × 3 × 3 mm3 voxels, b-value 500 seconds/mm2, and 90 different diffusion sensitizing gradient directions. Multivoxel tracts were generated along the vectors, corresponding to the directions of maximal diffusion in each voxel. The data was visualized using custom fiber tracking software and images compared with known anatomy.DTI tractography depicts the complete three-dimensional (3D) myoarchitecture of the human tongue, specifically demonstrating the geometric relationships between the intrinsic and extrinsic myofiber populations. These results define the manner in which key extrinsic fiber populations merge with the longitudinally-, transversely-, and vertically-aligned intrinsic fibers.The current results display for the first time the use of DTI tractography in vivo to visualize the complete structural anatomy of the human tongue and allow us to consider fundamental structure-function relationships.