Radial endobronchial ultrasound (R-EBUS) is one important diagnostic approach in non-small cell lung cancers (NSCLC). However, the small samples obtained from R-EBUS-guided transbronchial biopsies are sometimes insufficient for pathological and molecular diagnosis. Herein, we investigated the suitability of R-EBUS-guided bronchial brushing specimens for NSCLC diagnosis and EGFR genotyping. We enrolled 941 consecutive patients with peripheral pulmonary lesions who underwent R-EBUS. Cytology-positive brushing specimens from non-squamous NSCLC patients were tested for EGFR mutations. Non-squamous NSCLC was diagnosed in 624 patients (66.3%). Positive cytology was documented in the brushing samples of 376 patients (60.3%). Higher diagnostic yields were obtained in patients exhibiting bronchus signs on chest tomography, and those with R-EBUS probe located within the lesion. EGFR genotyping was successfully performed in 363 samples (96.5% of cytology-positive brushing samples). EGFR genotyping concordance between brushing specimens and matched tissue samples was 88.7% (kappa = 0.745, P < 0.001). Furthermore, 144 non-squamous NSCLC patients (23.1%) with failed pathological diagnosis or EGER sequencing by R-EBUS-guided transbronchial biopsy required repeat biopsies. However, it was achieved successfully from the brushing specimens of 57 patients (39.6%). In conclusion, for patients with peripheral lung cancer, R-EBUS-guided bronchial brushing could provide an additional sampling method for diagnosis and EGFR genotyping.