The biophysical and biomechanical properties of the crystalline lens (e.g., viscoelasticity) have long been implicated in accommodation and vision problems, such as presbyopia and cataracts. However, it has been difficult to measure such parameters noninvasively. Here, we used in vivo Brillouin optical microscopy to characterize material acoustic properties at GHz frequency and measure the longitudinal elastic moduli of lenses. We obtained three-dimensional elasticity maps of the lenses in live mice, which showed biomechanical heterogeneity in the cortex and nucleus of the lens with high spatial resolution. An in vivo longitudinal study of mice over a period of 2 months revealed a marked age-related stiffening of the lens nucleus. We found remarkably good correlation (log-log linear) between the Brillouin elastic modulus and the Young's modulus measured by conventional mechanical techniques at low frequencies (~1 Hz). Our results suggest that Brillouin microscopy is potentially useful for basic and animal research and clinical ophthalmology.