Characterization of myelin degradation in the white matter (WM) is important for understanding neurodegeneration. We demonstrate label-free in vivo imaging of myelin structure in the WM of mice, through intact cortex, using third harmonic generation (THG) microscopy at 1320-nm excitation. Longitudinal THG imaging of the same axons in the cuprizone mouse model of multiple sclerosis revealed dynamics of myelin blistering. Further, we measured intranodal distance at nodes of Ranvier in vivo and developed a novel metric of myelin structural change based on spatial concentration of the brightest THG signal. We also demonstrated compatibility with three-photon excited fluorescence microscopy by imaging GFP-labeled microglia in the WM in parallel with THG microscopy, thereby enabling detailed tracking of subcortical myelin and other cellular dynamics in neurodegenerative disease models.