INTRODUCTION: Immune checkpoint pathway markers induce immune tolerance to non-small cell lung cancer (NSCLC). Therapeutic antibodies targeting the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway have demonstrated efficacy in tumors expressing relatively high PD-L1 levels. Minimally invasive endobronchial ultrasound-guided fine needle aspiration allows patients with inoperable tumors or comorbidities to attain a confirmatory diagnosis. The aims of the present study were to determine whether PD-L1 testing is equivalent to cytology and biopsy or resection specimens at different tumor proportion score cutoffs and for different NSCLC subtypes. MATERIALS AND METHODS: Data were retrospectively collected for patients with paired NSCLC cytology and surgical resection specimens from May 4, 2007 to May 4, 2017. The Food and Drug Administration-approved Dako PD-L1 immunohistochemistry 22C3 pharmDx kit was used to measure PD-L1 on paired cytology cell block and biopsy or resection specimens, and the PD-L1 tumor proportion scores were recorded. Statistical analysis of categorical and continuous variables was performed using SAS, version 9.4. RESULTS: A total of 53 paired cytology and resection samples (27 adenocarcinoma, 25 squamous cell carcinoma, and 1 unclassified) were analyzed. Supposing the resection specimen to reflect the true PD-L1 expression, the sensitivity, specificity, positive predictive value, negative predictive value, and overall agreement for the cytology method was 73.3%, 65.2%, 73.3%, 65.2%, and 69.8%, respectively. For high PD-L1 expression (≥50%), the cytology method demonstrated an overall agreement of 79.2%. The overall agreement between methods was 81.5% and 76% for cases of adenocarcinoma and squamous cell carcinoma, respectively. CONCLUSIONS: NSCLC cytology samples from endobronchial ultrasound-guided fine needle aspiration are suitable for PD-L1 testing, especially using a high PD-L1 expression cutoff of ≥50% and for adenocarcinoma.