Knowledge of the normal myocardial-myocyte orientation could theoretically allow the definition of relevant quantitative biomarkers in clinical routine to diagnose heart pathologies. A whole heart diffusion tensor template representative of the global myofiber organization over species is therefore crucial for comparisons across populations. In this study, we developed a groupwise registration and tractography framework to resolve the global myofiber arrangement of large mammalian sheep hearts. To demonstrate the potential application of the proposed method, a novel description of sub-regions in the intraventricular septum is presented. Three explanted sheep (ovine) hearts (size ~12×8×6 cm3, heart weight ~ 150 g) were perfused with contrast agent and fixative and imaged in a 9.4T magnet. A group-wise registration of high-resolution anatomical and diffusion-weighted images were performed to generate anatomical and diffusion tensor templates. Diffusion tensor metrics (eigenvalues, eigenvectors, fractional anisotropy …) were computed to provide a quantitative and spatially-resolved analysis of cardiac microstructure. Then tractography was performed using deterministic and probabilistic algorithms and used for different purposes: i) Visualization of myofiber architecture, ii) Segmentation of sub-area depicting the same fiber organization, iii) Seeding and Tract Editing. Finally, dissection was performed to confirm the existence of macroscopic structures identified in the diffusion tensor template. The template creation takes advantage of high-resolution anatomical and diffusion-weighted images obtained at an isotropic resolution of 150 μm and 600 μm respectively, covering ventricles and atria and providing information on the normal myocardial architecture. The diffusion metric distributions from the template were found close to the one of the individual samples validating the registration procedure. Small new sub-regions exhibiting spatially sharp variations in fiber orientation close to the junctions of the septum and ventricles were identified. Each substructure was defined and represented using streamlines. The existence of a fiber-bundles in the posterior junction was validated by anatomical dissection. A complex structural organization of the anterior junction in comparison to the posterior junction was evidenced by the high-resolution acquisition. A new framework combining cardiac template generation and tractography was applied on the whole sheep heart. The framework can be used for anatomical investigation, characterization of microstructure and visualization of myofiber orientation across samples. Finally, a novel description of the ventricular junction in large mammalian sheep hearts was proposed.Read less <