Background: Antibody-based therapies for targeting programmed death 1 (PD-1) or its ligand PD-L1 (anti-PD1/PD-L1 therapies) have shown promising results in gastric cancer (GC). PD-L1 expression on tumor cell membrane and tumor-infiltrating lymphocytes (TILs) has been proposed as predictive biomarkers for anti-PD-1/PD-L1 therapies in several types of malignancies. TCGA has recently reported that PD-L1 gene was frequently amplified in Epstein-Barr virus (EBV)-positive GC. In addition, mismatch-repair deficiency (D-MMR) was associated with better clinical outcomes after anti-PD-1/PD-L1 therapies in solid tumors. However, little is known about clinicopathological features of PD-L1 expression with TILs, MMR and EBV status in a large cohort of GC cases. Methods: We performed a tissue microarray analysis in 487 advanced GC patients who underwent a gastrectomy without preoperative chemotherapy. PD-L1 expression on tumor cell membrane, densities as well as expressions of lymphocyte-associated markers (CD3, CD4, CD8, and FOXP3) of TILs and MMR status were evaluated by immunohistochemistry. EBV status was evaluated by in situ hybridization. Results: PD-L1 expression, D-MMR, and EBV were identified in 22.8, 5.1, and 5.1% of cases, respectively. PD-L1 expression was more frequently observed in elderly (P = 0.002), male (P = 0.029), poorly differentiated adenocarcinoma with solid-type histology (P < 0.001), D-MMR (P < 0.001), and EBV-positive status (P = 0.001). Regarding PD-L1 expression and TILs status, a strong association was observed between PD-L1 expression and high densities of either CD3 (+), CD8 (+), or FOXP3 (+) TILs (P < 0.001). In multivariate analysis, high density of CD8 (+) TILs was significantly associated with better survival (hazard ratio, 0.62; 95% CI, 0.39 to 0.99; P = 0.047), while PD-L1 expression and densities of other subtypes of TILs were not independent prognostic factors. Conclusions: In GC, PD-L1 expression was associated with distinct clinicopathological features including high density of TILs, D-MMR and EBV-positive status, but not a prognostic factor.