To explore the shape and the location of mediastinal spaces (mediastinal lymph node locations) associated with conventional transbronchial needle aspiration technique (C-TBNA), and to determine the relationship between the mediastinal spaces and physiological and anatomical marks in trachea-bronchial lumen for choosing the puncture points of C-TBNA. The chest HRCT images of 1 000 consecutive patients preparing for bronchoscopic examination were analyzed to determine the shape and the location of the mediastinal gap, and which physiological and anatomic markers in the airway were suitable for locating the mediastinal spaces and lymph nodes. Eighty-one groups of lymph nodes from 52 patients were punctured by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to verify the locating of puncture points by these markers for the mediastinal lymph nodes under general anesthesia. The needle was punctured into the wall of the airway first, and then the ultrasound was used to confirm if the needle was in the lymph nodes. Pretracheal space, aortic-left pulmonary window space, superior-carinal space and subcarinal space showed close relationship with C-TBNA biopsy for mediastinal lymph nodes. The pretracheal space could be located by the physiological and anatomic marks of aortic impression and aortic pulse points. The superior-carinal space was like a triangle and locating in 12 o'clock of the first ring of trachea. The 9-10 o'clock of the first ring of the trachea was the aortic-left pulmonary window. The 8-9 o'clock of the right main bronchus and middle bronchus was for subcarinal space. Eighty-one groups of lymph nodes from 52 patients were punctured by EBUS-TBNA according to the physiological and anatomic markers, and it showed that only 3 groups were missed by C-TBNA. No mediastinal bleeding and pneumomediastinum occurred. Mediastinal spaces(mediastianl lymph nodes) and physiological and anatomical marks within the airway had a relatively fixed relationship. These marks could be used for locating the mediastinal lymph nodes when C-TBNA was performed.