Ex vivo assessment of microscale tissue biomechanical properties of the mammalian retina could offer insights into diseases such as keratoconus, and macular degeneration. A 250-MHz scanning acoustic microscope (7-μm resolution) has been constructed to derive two-dimensional quantitative maps of attenuation (α), speed of sound (c), acoustic impedance (Z), bulk modulus (B), and mass density ( ρ). The two-dimensional maps were compared to coregistered hematoxylin-and-eosin stained sections. This study is the first to quantitatively assess α, c, Z, B, and ρ of individual retinal layers of mammalian animals at high ultrasound frequencies. Significant differences in these parameters between the layers were demonstrated.