All existing constitutive models for heart valve leaflet tissues either assume a uniform transmural stress distribution or utilize a membrane tension formulation. Both approaches ignore layer specific mechanical contributions and the implicit nonuniformity of the transmural stress distribution. To begin to address these limitations, we conducted novel studies to quantify the biaxial mechanical behavior of the two structurally distinct, load bearing aortic valve (AV) leaflet layers: the fibrosa and ventricularis. Strip biaxial tests, with extremely sensitive force sensing capabilities, were further utilized to determine the mechanical behavior of the separated ventricularis layer at very low stress levels. Results indicated that both layers exhibited very different nonlinear, highly anisotropic mechanical behaviors. While the leaflet tissue mechanical response was dominated by the fibrosa layer, the ventricularis contributed double the amount of the fibrosa to the total radial tension and experienced four times the stress level. The strip biaxial test results further indicated that the ventricularis exhibited substantial anisotropic mechanical properties at very low stress levels. This result suggested that for all strain levels, the ventricularis layer is dominated by circumferentially oriented collagen fibers, and the initial loading phase of this layer cannot be modeled as an isotropic material. Histological-based thickness measurements indicated that the fibrosa and ventricularis constitute 41% and 29% of the total layer thickness, respectively. Moreover, the extensive network of interlayer connections and identical strains under biaxial loading in the intact state suggests that these layers are tightly bonded. In addition to advancing our knowledge of the subtle but important mechanical properties of the AV leaflet, this study provided a comprehensive database required for the development of a true 3D stress constitutive model for the native AV leaflet.