Objective. Optical coherence tomography (OCT), an imaging technology that provides cross-sectional subsurface tissue structure images using back-scattered light, is a promising noninvasive imaging modality for in vivo assessment of vocal fold layered microstructure. Polarization-sensitive OCT (PS-OCT) augments conventional OCT by detecting changes in the polarization state of reflected light. This study imaged various benign and malignant laryngeal pathologies in patients undergoing direct laryngoscopy under general anesthesia to determine whether PS-OCT would provide useful additional information about vocal fold microstructure and glottic surface pathology. Study Design. Prospective clinical trial. Setting. Tertiary care center. Subjects and Methods. Twenty-three patients who were undergoing microlaryngoscopy under general anesthesia for benign (n = 18) and malignant (n = 5) glottic disease were imaged bilaterally with OCT and PS-OCT (n = 46 vocal folds). Intraoperative microphotography guided placement of the imaging probe. Normal-appearing glottic tissue was also imaged if present. When clinically indicated, biopsy or complete removal of the lesion established histologic confirmation. Results. PS-OCT provided high-quality, vertical, cross-sectional images up to 1.2-mm deep that complemented microlaryngoscopy and conventional OCT for vocal fold pathologies. Scar tissue was visualized by PS-OCT, characterized by a birefringence pattern more intense than that of normal glottic tissue. Cancer typically showed disruption or absence of both layered structure and birefringence. Conclusion. Combining PS-OCT with OCT during human vocal cord imaging provides useful information in characterizing vocal cord lesions, particularly scar tissue. © American Academy of Otolaryngology-Head and Neck Surgery Foundation 2011.