Demyelination is the hallmark of some neurodegenerative autoimmune diseases, such as multiple sclerosis and Guillain–Barr� syndrome. To understand the mechanism of myelin degradation and monitor the outcome of remyelinating therapies, real-time visualization of myelin changes under certain condition at the resolution of single cell level is desired. This chapter introduces coherent anti-Stokes Raman scattering (CARS) microscopy which allows label-free molecular imaging of myelin in its natural state at the submicron resolution. The principle of CARS and the key components of a CARS microscope are described. Step-by-step procedures for CARS imaging of myelin in fresh tissues, fixed slices, cryosections, and live animals are provided. Finally, new findings of demyelination mechanism revealed by CARS microscopy are discussed.