Background The efficacy of transbronchial needle aspiration (TBNA) with endobronchial ultrasonography using a guide sheath (EBUS-GS) for cases of peripheral pulmonary lesions (PPLs) has not been well established. The purpose of this study was to evaluate the efficacy of TBNA with EBUS-GS for PPLs. Methods We evaluated 130 patients suspected to have lung cancer who underwent transbronchial brushing (brushing), transbronchial biopsy (TBB), and TBNA with EBUS-GS. The pathological diagnostic yields of TBNA were compared to that of TBB and brushing. The histological diagnosis of TBNA was compared to that of surgical specimens. The results of epidermal growth factor receptor (EGFR) gene mutation in TBNA samples were compared to that in TBB or surgical specimens. Results The diagnostic yields of this study were 62.9% for brushing, 80.0% for TBB, and 77.1% for TBNA. Histological diagnosis was 84.8% for TBB and/or TBNA and pathological diagnosis was 86.7% for all the procedures. TBB and TBNA had significance higher than brushing (p &lt
0.05). TBB and TBNA had a tendency of higher diagnostic yields than brushing if EBUS probe was adjacent to PPLs (p = 0.058). Histological evaluations were obtained from TBNA specimens from 50 of 105 patients (47.6%) and these were identical to those of surgical specimens from 29 of 32 patients (90.6%). The results of EGFR gene mutation in TBNA specimens were identical to the same tissue obtained by surgery or TBB. Conclusions TBNA with EBUS-GS for PPLs was a useful tool for accurate diagnosis and EGFR gene mutation analysis. This method may improve diagnostic accuracy and be useful for molecular testing. This study was approved by the institutional review board (Date of approval: May 27, 2013, approval number: 2816) of Tokyo Women's Medical University Hospital.