Accurate wavelength assignment of each spectral element for spectral-domain optical coherence tomography (SD-OCT) and optical frequency domain imaging (OFDI) is required for proper construction of biological tissue cross-sectional images. This becomes more critical for functional extensions of these techniques, especially in polarization-sensitive optical coherence tomography (PS-OCT), where incorrect wavelength assignment between the two orthogonal polarization channels leads to polarization artifacts. We present an autocalibration method for wavelength assignment that does not require separate calibration measurements and that can be applied directly on actual data. Removal of the birefringence artifact is demonstrated in a PS-OCT system with picometer accuracy in the relative wavelength assignment, resulting in a residual phase error of . We also demonstrate, for the first time, a quantitative birefringence map of an in vivo human retinal nerve fiber layer.