Background: Optical coherence tomography (OCT) is a new technology capable of generating high resolution cross-sectional images of complex tissue in real time. Analogous to ultrasound, OCT measures backscattered light intensity using coherence interferometery to construct topographical images of complex tissue. Since OCT uses infrared light rather than acoustic waves, its spatial resolution is exceptionally high (2–10 µm). Recent advances in data acquisition, analysis, and processing enable real-time imaging, and make OCT a potentially valuable tool for pulmonary airway diagnostic applications, including assisting directed airway biopsies. Objective: This study evaluates feasibility of OCT for delineating proximal airway microstructures in various animal as well as human tracheas. Methods: Excised trachea samples from New Zealand white rabbits, Duroc pigs, and human trachea were imaged using a compact, 1,300-nm broad-band superluminescent-diode-based prototype fiber OCT device we constructed. The resulting structural OCT images were compared to conventional hematoxilin and eosin (HE) stained histological sections from the same samples. Results: OCT was able to delineate microstructures such as the epithelium, mucosa, cartilage, and glands in all samples. Conclusion: These findings suggest that integration of OCT with flexible fiberoptic bronchoscopy could enhance pulmonary diagnostic medicine and detection of pathologic tissue changes in various respiratory diseases.