Lung cancer is the leading cause of cancer death in the United States, accounting for 28% of all cancer deaths. Standard of care for potentially curable lung cancer involves preoperative radiographic or invasive staging, followed by surgical resection. With recent adjuvant chemotherapy and radiation studies showing a survival advantage in nodepositive patients, it is crucial to accurately stage these patients surgically in order to identify those who may benefit. However, lymphadenectomy in lung cancer is currently performed without guidance, mainly due to the lack of tools permitting real-time, intraoperative identification of lymph nodes. In this study we report the design and validation of a novel, clinically compatible near-infrared (NIR) fluorescence thoracoscope for real-time intraoperative guidance during lymphadenectomy. A novel, NIR-compatible, clinical rigid endoscope has been designed and fabricated, and coupled to a custom source and a dual channel camera to provide simultaneous color and NIR fluorescence information to the surgeon. The device has been successfully used in conjunction with a safe, FDA-approved fluorescent tracer to detect and resect mediastinal lymph nodes during thoracic surgery on Yorkshire pigs. Taken together, this study lays the foundation for the clinical translation of endoscopic NIR fluorescence intraoperative guidance and has the potential to profoundly impact the management of lung cancer patients.