Objective To evaluate the role of guide sheath (GS) utilization in radial endobronchial ultrasound guided transbronchial lung biopsy (EBUS-TBLB) for diagnosis of peripheral pulmonary lesions (PPLs). Methods The clinical data of patients who underwent EBUS-TBLB in Peking University First Hospital from July 2012 to June 2015 were retrospectively reviewed. The patients were divided into three groups,ie. a GS group, a non-GS group, and a double biopsy group. Results A total of 118 patients with 126 PPLs were collected. The overall diagnostic yield of EBUS-guided bronchoscopy was 60.3%. The diagnostic yield of GS group, non-GS group and double biopsy group was 65.4%(36/55), 61.5%(8/13), 59.6%(31/52), respectively. The diagnostic yield of the non-GS group was significantly lower than other two groups when PPLs≤20 mm (χ2=6.8,P=0.033), whereas no significant difference was observed when PPLs>20 mm (χ2=2.301,P=0.301). Conclusion GS significantly improves diagnostic yield in EBUS-TBLB when PPLs≤20 mm.