Knowledge of temperature-induced changes of skin optical properties is required for accurate dosimetry of photothermal treatments. We determined and compared in vivo optical properties of mouse ear skin at different temperatures. The diffuse reflectance, total and diffuse transmittance were measured in the spectral range from 400 to 1650 nm using an integrating sphere spectrometer at the temperatures of 25 °C, 36 °C and 60 °C. Target temperatures were attained and maintained using an automated heater equipped with a sensor for feed-back and control. Temperature and temperature induced morphological changes of skin were monitored using an infrared thermal camera and reflectance confocal microscopy, respectively. An inverse Monte Carlo technique was utilized to determine absorption, scattering, and anisotropy factors from the measured quantities. Our results indicate significant differences between the optical properties of skin at different temperatures. Absorption and scattering coefficients increased, whereas anisotropy factors decreased with increasing temperature. Changes in absorption coefficients indicate deoxygenation of hemoglobin, and a blue shift of water absorption bands. Confocal imaging confirmed that our observations can be explained by temperature induced protein denaturation and blood coagulation. Monitoring spectral responses of treated tissue may become a valuable tool for accurate dosimetry of light treatments.