The myocardium consists of a complex 3-dimensional (3D) microstructure has been shown to be perturbed in the presence of myocardial ischemia. Recently, diffusion tensor magnetic resonance imaging (DT-CMR) was introduced which can characterize the 3D tissue microstructure in intact myocardium. However, past histologic validation of DTI has been limited since traditional pathology allows only 2D optical microscopy after potentially destructive tissue sectioning. We present a novel approach to validate the derivation of the myocardial fiber orientation (MFO) using DT-CMR with 3D histology using a non-destructive, transparent-tissue preparation technique (CLARITY). Results indicate MFO derived from 3D histology and DT-CMR are strongly concordant.