Although myocardial architecture has been investigated extensively, as yet no evidence exists for the anatomic segregation of discrete myocardial pathways. We performed post-mortem diffusion tensor imaging on 14 pig hearts. Pathway tracking was done from 22 standardized voxel groups from within the left ventricle, the left ventricular papillary muscles, and the right ventricular outflow tract. We generated pathways with comparable patterns in the different hearts when tracking from all chosen voxels. We were unable to demonstrate discrete circular or longitudinal pathways, nor to trace any solitary tract of myocardial cells extending throughout the ventricular mass. Instead, each pathway possessed endocardial, midwall, and epicardial components, merging one into another in consistent fashion. Endocardial tracks, when followed towards the basal or apical parts of the left ventricle, changed smoothly their helical and transmural angulations, becoming continuous with circular pathways in the midwall, these circular tracks further transforming into epicardial tracks, again by smooth change of the helical and transmural angles. Tracks originating from voxels in the papillary muscles behaved similarly to endocardial tracks. This is the first study to show myocardial pathways that run through the mammalian left and right ventricles in a highly reproducible manner according to varying local helical and transmural intrusion angles. The patterns generated are an inherent feature of the three-dimensional arrangement of the individual myocytes aggregated within the walls, differing according to the regional orientation and branching of individual myocytes. We found no evidence to support the existence of individual muscles or bands. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.