The anatomy of the mammalian tongue consists of an intricate array of variably aligned and extensively interwoven muscle fibers. As a result, it is particularly difficult to resolve the relationship between the tongue's microscopic anatomy and tissue-scale mechanical function. In order to address this question, we employed a method, diffusion spectrum imaging (DSI) with tractography, for displaying the macroscopic orientational properties of the tissue's constituting myofibers. DSI measures spatially variant proton displacement for a given 3D imaging segment (voxel), reflecting the principal orientation(s) of its myofibers. Tractography uses the angular similarity displayed by the principal fiber populations of multiple adjacent voxels to generate tract-like structures. DSI with tractography thus defines a unique set of tracts based on the net orientational behavior of the myofiber populations at different positions in the tissue. By this approach, we demonstrate a novel myoarchitectural pattern for the bovine tongue, consisting of short and orthogonally aligned crossing fiber tracts in the intrinsic core region, and longer, parallel-aligned fiber tracts on the tissue margins and in the regions of extrinsic fiber insertion. The identification of locally aligned myofiber populations by DSI with tractography allows us to reconsider lingual anatomy, not in conventional microscopic terms, but as a set of heterogeneously aligned and macroscopically resolved myofiber tracts. We postulate that the properties associated with these myofiber tracts predict the mechanical behavior of the tissue and thus constitute a method to relate structure and function for anatomically complex muscular tissues. Anat Rec Part A, 288A:1173–1182, 2006. © 2006 Wiley-Liss, Inc.