We have recently proposed an optical method for assessing heart structure that uses polarized light measurement of birefringence as an indicator of tissue anisotropy. The highly aligned nature of healthy cardiac muscle tissue has a detectable effect on the polarization of light, resulting in a measurable phase shift (“retardance”). When this organized tissue structure is perturbed, for example after cardiac infarction (heart attack), scar tissue containing disorganized collagen is formed, causing a decrease in the measured retardance values. However, these are dependent not only on tissue anisotropy, but also on the angle between the tissue’s optical anisotropy direction and the beam interrogating the sample. To remove this experimental ambiguity, we present a method that interrogates the sample at two different incident beam angles, thus yielding enough information to uniquely determine the true magnitude and orientation of the tissue optical anisotropy. We use an infarcted porcine heart model to compare these polarimetryderived anisotropy metrics with those obtained with diffusion tensor magnetic resonance imaging (DT-MRI). The latter yields the anisotropy and the direction of tissue water diffusivity, providing an independent measure of tissue anisotropy. The optical and MR results are thus directly compared in a common ex vivo biological model of interest, yielding reasonable agreement but also highlighting some technique-specific differences.