Polarization-sensitive optical coherence tomography (PS-OCT) is an augmented form of OCT, providing 3D images of both tissue structure and polarization properties. We developed a new method of polarization-sensitive optical frequency domain imaging (PS-OFDI), which is based on a wavelength-swept source. In this method the sample was illuminated with unpolarized light, which was composed of two orthogonal polarization states (i.e., separated by 180° in the Poincaré sphere) that are uncorrelated to each other. Reflection of these polarization states from within the sample was detected simultaneously and independently using a frequency multiplexing scheme. This simultaneous sample probing with two polarization states enabled determination of the depth-resolved Jones matrices of the sample. Polarization properties of the sample were obtained by analyzing the sample Jones matrices through eigenvector decomposition. The new PS-OFDI system ran at 31K wavelength-scans/s with 3072 pixels per wavelength-scan, and was tested by imaging a polarizer and several birefringent tissues such as chicken muscle and human skin. Lastly the new PS-OFDI was applied to imaging two cancer animal models: a mouse model by injecting cancer cells and a hamster cheek pouch model. These animal model studies demonstrated the significant differences in tissue polarization properties between cancer and normal tissues in vivo. © 2011 Optical Society of America.