Background: Gastric cancer (GC) is a deadly disease with limited treatment options. Recent studies with PD-1 inhibitor Pembrolizumab have shown promising results in GC. Key questions remain which GC subclass may respond best. In a comprehensive molecular analysis of gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) we identified that 15% of Epstein Barr Virus (EBV)+ GCs have amplification of chromosomal region 9p24.1, the locus of PD-1 ligands PD-L1 and PD-L2. As EBV+ cancers have multiple mechanisms to induce PD-L1 expression, we hypothesize that PD-L1 status may differ specifically in EBV+ GCs, regardless of 9p24.1 status, indicating a broader potential role for PD-1 blockade in this GC subtype. Methods: PD-L1 expression was analyzed with immunohistochemistry (IHC) using whole tissues slides of EBV+ (n = 32) and EBV negative (n = 49) GCs. The transcriptional landscape of EBV+ and EBV negative GCs was determined using mRNA sequencing results from the TCGA study. Results: With IHC we showed that EBV+ GCs have PD-L1 expression in tumor and immune cells in 50% (16/32) and 94% (30/32) of GCs respectively. This in contrast to EBV negative GCs that have PD-L1+ tumor and immune cells in only 10% (5/49) and 39% (19/49) of cases respectively (P< 0.001). Furthermore, PD-L1+ immune cells infiltrated the center of the majority of EBV+ GCs, while in EBV negative GCs PD-L1+ immune cells remained at the invasive margin (P = 0.007). mRNA sequencing analyses of 12 EBV+ GCs and 10 EBV- GCs showed that PD-L1+/EBV+ GCs have enrichment of an interferon-γ driven gene signature. Single-sample gene set enrichment analyses on a bigger series of 214 individual GCs demonstrated that besides EBV+ GCs (n = 19), microsatellite instable (MSI) GCs (n = 47) have strong enrichment of IFN-γ response gene expression, in contrast to genomic stable (GS) (n = 42) and chromosomal instable (CIN) (n = 106) gastric cancer subtypes (P< 0.001). Conclusions: As the presence of PD-L1+ tumor and/or tumor infiltrating immune cells as well as an interferon-mediated adaptive immune response are associated with PD-1 blockade vulnerability in other cancers, we here provide a strong rationale for testing of PD-1 blockade in EBV+ GC and for evaluating EBV and MSI status as variables for response.