Background: Pembrolizumab and atezolizumab monotherapy has been the standard of care in treatment-naïve advanced NSCLC with high PD-L1 expression. On the other hand, the evaluation of PD-L1 expression as companion diagnostics is different between two agents (Clone: 22C3 and SP142). PD-L1 Immunohistochemistry Comparability Study showed that the SP142 assay showed significantly less sensitivity to detect PD-L1 expression on TCs, leading to the discrepancy in cases with PD-L1 high expression on tumor cells (TCs). This study aimed to evaluate the clinical impact of the discrepancy in PD-L1 high expression on the efficacy of pembrolizumab monotherapy in advanced NSCLC patients. Methods: We retrospectively reviewed advanced NSCLC patients with PD-L1 high expression (22C3: TPS ≥ 50%) who received first-line pembrolizumab monotherapy, and had sufficient tumor tissues for evaluating PD-L1 expression on TCs (SP142). Additionally, RNA-sequence analysis was performed to evaluate the biological differences in the discrepancy cases with PD-L1 high expression on TCs. Results: A total of 45 patients (PD-L1 expression TPS [22C3]: ≥ 50%) were included. Of these patients, 5 / 9 / 18 / 13 patients had PD-L1 expression (SP142): TC 0 / 1 / 2 / 3, respectively. The objective response rate (ORR) and median PFS (mPFS) of pembrolizumab in all pts were 62% (95% confidence interval [CI], 47-76) and 7.52 months (mo, 95% CI, 4.73-10.97). The ORR and PFS in patients with TC ≤ 1 were significantly worse than those with TC ≥ 2 (TC ≤ 1 vs. TC ≥ 2: ORR 21% vs. 81%, p < 0.001, and mPFS 3.81 mo vs. 8.05 mo, p = 0.004, Table). In patients with PD-L1 TPS ≥ 90% (n = 34), the ORR and PFS in patients with TC ≤ 1 were also significantly worse than those with TC ≥ 2 (TC ≤ 1 vs TC ≥ 2: ORR 0% vs. 82%, p < 0.001, and mPFS 2.04 mo vs. 10.97 mo, p < 0.001, Table). Additionally, RNA-sequence analysis showed that tumors with TC ≤ 1 had significantly suppressed immune-related pathways (ALLOGRAFT REJECTION and IFNG RESPONSE) and significantly lower T-cell-inflamed gene expression profiles compared with those with TC ≥ 2. Conclusions: Discrepancy in PD-L1 expression on tumor cells between 22C3 and SP142 assay was a negative predictive factor for pembrolizumab monotherapy and showed suppressed tumor microenvironment.