The myoarchitecture of the ventricular wall provides a structural template dictating tissue-scale patterns of mechanical function. We studied whether myofiber tract imaging performed with MR diffusion spectrum imaging (DSI) tractography has the capacity to resolve abnormalities of ventricular myoarchitecture in a model of congenital hypertrophic cardiomyopathy (HCM) associated with the ablation of myosin binding protein-C (MyBP-C). Homozygous MyBP-C knockout mice were generated by deletion of exons 3-10 from the endogenous MyBP-C gene. Fiber alignment in the left ventricular wall of wild type mice was depicted through DSI tractography (and confirmed by multi-slice two-photon microscopy) as a set of helical structures whose angles display a continuous transition from negative in the subepicardium to positive in the subendocardium. In contrast, the hearts obtained from the MyBP-C knockouts displayed substantial myoarchitectural disarray, characterized by a loss of voxel-to-voxel orientational coherence for fibers principally located in the mid-myocardium-subendocardium and impairment of the transmural progression of helix angles. These results substantiate the use of DSI tractography in determining myoarchitectural disarray in models of cardiomyopathy and suggest a biological association between myofilament expression, cardiac fiber alignment, and torsional rotation in the setting of congenital HCM.