Diffusion tensor magnetic resonance imaging (MRI) is a possible new means of elucidating the anatomic structure of the myocardium. It enjoys several advantages over traditional histological approaches, including the ability to rapidly measure fiber organization in isolated, perfused, arrested hearts, thereby avoiding fixation and sectioning of artifacts. However, quantitative validation of this MRI method has been lacking. Here, fiber orientations estimated in the same locations in the same heart using both diffusion tensor MRI and histology are compared in a total of two perfused rabbit hearts. Fiber orientations were statistically similar for both methods and differed on average by 12 degrees at any single location. This is similar to the 10 degrees uncertainty in fiber orientation achieved with histology. In addition, imaging studies performed in a total of seven hearts support a level of organization beyond the myofiber, the recently described laminar organization of the ventricular myocardium.