A 64-year-old man with back pain was admitted to our hospital. Magnetic resonance imaging (MRI) showed spine metastases, and positron emission tomography (PET) showed a right hilar and mediastinal lymph node and spine accumulation. An endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the right hilar lymph node showed malignant cells. As a result, we ultimately diagnosed cancer of unknown primary (CUP). The autopsy revealed metastases extending to multiple organs, whereas the primary lesion of cancer was not detected. The diagnosis of CUP is difficult because of multiple metastases. We encountered a case in which we successfully diagnosed CUP by EBUS-TBNA with minimum invasion and verified the diagnosis by autopsy. The EBUS-TBNA is useful for the diagnosis of CUP when the metastases of hilar and mediastinal lymph nodes were suspected.