We report on application of pulsed photothermal radiometry (PPTR) to determine the depth of port wine stain (PWS) blood vessels in human skin. When blood vessels are deep in the PWS skin (>100 microm), conventional PPTR depth profiling can be used to determine PWS depth with sufficient accuracy. When blood vessels are close or partially overlap the epidermal melanin layer, a modified PPTR technique using two-wavelength (585 and 600 nm) excitation is a superior method to determine PWS depth. A direct difference approach in which PWS depth is determined from a weighted difference of temperature profiles reconstructed independently from two-wavelength excitation is demonstrated to be appropriate for a wider range of PWS patients with various blood volume fractions, blood vessel sizes, and depth distribution. The most superficial PWS depths determined in vivo by PPTR are in good agreement with those measured using optical Doppler tomography (ODT).