Anisotropy of mouse and human skin is investigated in vivo using polarized videoreflectometry. An incident beam (linearly polarized, wavelength 650 nm) is focused at the sample surface. Two types of tissuelike media are used as controls to verify the technique: isotropic delrin and highly anisotropic demineralized bone with a priori knowledge of preferential orientation of collagen fibers. Equi-intensity profiles of light, backscattered from the sample, are fitted with ellipses that appear to follow the orientation of the collagen fibers. The ratio of the ellipse semiaxes is well correlated with the ratio of reduced scattering coefficients obtained from radial intensity distributions. Variation of equi-intensity profiles with distance from the incident beam is analyzed for different initial polarization states of the light and the relative orientation of polarization filters for incident and backscattered light. For the anisotropic media (demineralized bone and human and mouse skin), a qualitative difference between intensity distributions for cross- and co-polarized orientations of the polarization analyzer is observed up to a distance of 1.5 to 2.5 mm from the entry point. The polarized videoreflectometry of the skin may be a useful tool to assess skin fibrosis resulting from radiation treatment. © 2005 Society of Photo-Optical Instrumentation Engineers.