Abstract Background The diagnostic yield of peripheral pulmonary lesions has significantly increased with the use of radial endobronchial ultrasound with guide sheath within the lesion. Here, we retrospectively evaluated factors leading to misdiagnosis of pulmonary malignant tumors using endobronchial ultrasound with the guide sheath within the lesion. Methods We assessed the final histopathological diagnosis of biopsy samples taken from 130 patients with lung malignant tumors that underwent endobronchial ultrasound with guide sheath within the lesion. Results Among 130 patients, 8 (6%) showed no definite malignant findings in biopsy samples but the presence of malignant cells (primary lung cancer 7, diffuse large B cell lymphoma 1) was subsequently confirmed by histopathological study of specimens taken by computed tomography-guided needle biopsy or surgery. Of the eight cases with diagnostic failure, the size of the biopsy sample was insufficient in five due to technical difficulties during the diagnostic procedure, and the diagnosis of malignant tumor was difficult in five cases because of extensive scarring tissue or central necrosis. Conclusions The results of this study showed that technical difficulties and/or pathological heterogeneity of the tumor might lead to failure to diagnose lung malignant tumor in cases using endobronchial ultrasound with guide sheath within the lesion.