Simple SummaryEpstein–Barr virus (EBV) is associated with nearly 10% of all gastric cancers (GCs). GCs associated with EBV infection (EBVaGCs) are distinct from EBV-negative GCs (EBVnGCs) in terms of clinical outcomes, histology, and molecular features, including gene expression and methylation. While the continued expression of viral genes in EBVaGCs contributes to the carcinogenesis process, viral proteins also represent foreign antigens that could trigger immune responses that may greatly differ from EBVnGCs. The molecular differences in the tumor microenvironment between EBVaGCs and EBVnGCs have not been well characterized. We explored the differences in cell populations between EBVaGC and EBVnGC using a combination of bulk tumor and single-cell RNA sequencing data. We identified three unique immune cell subpopulations in EBVaGC: actively dividing T and B cells and B cells that also expressed T cell features. The less proliferative T cell cluster and the carcinoma cells from EBVaGC also exhibited unique features indicative of increased inflammation.AbstractEpstein–Barr virus (EBV) is a gamma-herpesvirus associated with nearly 10% of gastric cancers (GCs). These EBV-associated GCs (EBVaGCs) are molecularly, histopathologically, and clinically distinct from EBV-negative GCs (EBVnGCs). While viral genes in EBVaGCs contribute to the carcinogenesis process, viral proteins also represent foreign antigens that could trigger enhanced immune responses compared to EBVnGCs. Despite prior investigations of the EBVaGC tumor microenvironment (TME), the cellular composition has not been thoroughly explored. In this study, cellular subpopulations overrepresented in EBVaGCs were identified and molecularly characterized. Genes consistently expressed across both bulk tumor and single-cell RNA sequencing data were highlighted, with the expression across the identified cellular subpopulations analyzed. As expected, based on existing histopathological analysis, EBVaGC is characterized by abundant lymphocytic infiltration of the stroma. Our molecular analysis identified three unique immune cell subpopulations in EBVaGC: T and B cells expressing high levels of proliferation markers and B cells expressing T cell features. The proliferating T cell cluster also expressed markers of follicular T helper cells. Overall, EBVaGC also exhibited unique features indicative of a higher inflammatory response. These substantial differences within the TME suggest that further detailed exploration of the cellular composition of EBVaGCs is needed, which may identify cellular subpopulations and phenotypes associated with patient outcomes.