The diagnosis of peribronchial pulmonary lesions located in the tertiary bronchi, also known as segmental bronchi, as well as, the 4th order and 5th order segmental bronchi is very difficult. Histopathological specimens cannot be easily obtained by endobronchial biopsies (EBBX) due to the patent but small segmental bronchial lumen. The aim of the present study was to evaluate the diagnostic accuracy and safety of the novel technique with radial probe endobronchial ultrasound (R-EBUS) assisted conventional transbronchial needle aspiration (C-TBNA) in the diagnosis of solitary peribronchial pulmonary lesions located in segmental bronchi from 3th to 5th order. From December 2014 to December 2015, 16 patients with solitary peribronchial pulmonary lesions in the segmental bronchi from 3th to 5th order confirmed by computed tomography (CT) were enrolled. The lesions were located using radial probe endobronchial ultrasound (R-EBUS) to determine the sites of conventional transbronchial needle aspiration (C-TBNA), then, histopathological specimens were obtained using the technique of C-TBNA. The final pathological diagnosis was made based on the findings from the surgical specimens. Statistical analyses were performed for specimen results and complications. On pathological evaluation, 14 of the 16 specimens were malignant, including 8 adenocarcinomas, 4 squamous cell carcinomas, and 2 small cell carcinomas, while 2 were non-malignant diseases. The diagnostic accuracy rate, sensitivity and missed diagnosis rates were 87.5%, 87.5% and 12.5%, respectively. When Combined the results of cytology with histologic samples obtained from C-TBNA the total diagnostic accuracy rate, sensitivity and missed diagnosis rate were 93.75%, 93.75% and 6.25%, respectively. There were 2 cases of bleeding complications >5 mL after C-TBNA, and both were resolved with endobronchial management. The combination of R-EBUS with C-TBNA was advantageous and safe for the diagnosis of solitary peribronchial pulmonary lesions located in the segmental bronchi. However, possible bleeding complications should be anticipated with needle aspiration. Further verification of this combined application should be investigated in larger clinical trials.