Despite the frequent failure of aortic valves and pediatric usage of pulmonary valves as a replacement, comparative studies on their full-field collagenous architecture and macroscale geometries are limited. We applied laser micrometry and quantitative polarized light imaging (QPLI), a novel technique for assessing collagen fiber organization, to porcine aortic (n = 8) and pulmonary (n = 8) valve leaflets to non-destructively compare thickness and anisotropy. We confirmed (1) light intensity and sample thickness are inversely related; and (2) aortic valve leaflets are thicker with decreased fiber organization when unloaded. To demonstrate the ability of QPLI to capture dynamic collagen fiber alignment, we imaged leaflets during equibiaxial loading. There was an increase in the aortic valve leaflet’s degree of alignment throughout loading, whereas the pulmonary valve leaflet exhibited relatively unchanged alignment. Understanding the full-field organization of a leaflet’s heterogeneous ECM and how it is altered by pathology can inform therapy development.