Background <p>We compared systematic endobronchial ultrasound-guided transbronchial needle aspiration (S-EBUS-TBNA) [sampling every lymph node (LN) &gt; 5 mm regardless of its appearance on positron emission tomography/computed tomography (PET/CT)] with targeted (T) EBUS-TBNA (sampling only abnormal LNs on PET/CT) for mediastinal staging of locally advanced non-small cell lung cancer (NSCLC).</p> Patients and Methods <p>Patients with NSCLC with N2 involvement on PET/CT who underwent S-EBUS-TBNA were retrospectively included. For T-EBUS-TBNA, the results of the samplings of abnormal PET/CT LNs during S-EBUS-TBNA were considered. The percentage of cases where S-EBUS-TBNA diagnosed a larger extent of mediastinal disease compared with T-EBUS-TBNA (upstaging from N2a to N2b/N3 and upstaging from N2b to N3) was estimated.</p> Results <p>A total of 89 patients were included: 61 had N2a and 28 had N2b on PET/CT. Of the 61 with N2a S-EBUS-TBNA diagnosed N3 disease in 2 cases, N2b in 4, N2a in 43, and N0/1 in 12. These 12 patients underwent video-assisted mediastinoscopy (VAM) that showed N2a involvement in 3 and N0/1 in 9. Of the 28 with N2b, S-EBUS-TBNA proved N3 disease in 2 cases, N2b in 10 cases, N2a in 7, and N0/1 in 9. These nine patients underwent confirmatory VAM that showed N2a in two and N0/1 in 7. S-EBUS-TBNA diagnosed a larger extent of mediastinal disease compared with T-EBUS-TBNA staging in 9% of cases: 4 patients with N3 disease that had N2 on PET/CT and four with N2b that had N2a on PET/CT.</p> Conclusions <p>In patients with NSCLC, S-EBUS-TBNA diagnoses a larger extent of mediastinal disease compared with T-EBUS-TBNA.</p>