Background: Recently remarkable efficacy of immune checkpoint inhibition has been reported for several kinds of solid cancers. This study is the first comprehensive analysis of cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 ligand 1 (PD-L1) in gastric cancer and the first study integrating oncogenomic analyses. Methods: PD-L1 and CTLA-4 were stained on paraffin embedded tumor sections of 127 patients with gastric cancer by immunohistochemistry. Genetic driver mutations were identified by next-generation Sequencing and FISH analysis. Expression of PD-1, PD-L1 and CTLA-4 on lymphocytes in tumor sections, lymph nodes and peripheral blood were studied by 10-colour flow cytometry and 4-colour immune-fluorescence microscopy in an additional cohort. Results: PD-L1 and CTLA-4 were expressed on primary tumor cells by 44.9%(57/127) and 86.6%(110/127) of the analyzed gastric cancer samples, respectively. Correlation to clinical and pathological parameters revealed no correlation for PD-L1, whereas CTLA-4-negativity was correlated to higher grading and diffuse type according to Lauren. Positivity of PD-L1 or CTLA-4 on tumor cells was associated with inferior overall survival. Expression of PD-1 (52.2%), PD-L1 (42.2%) and CTLA-4 (1.6%) on tumor-infiltrating T cells was significantly elevated compared to peripheral blood lymphocytes. We could identify distinct genotypic profiles comparing the subgroups of checkpoint molecule expression. Conclusions: Our analysis revealed a great impact of PD-1/PD-L1 and CTLA-4 on the biology of gastric cancer. Hence corresponding checkpoint-inhibitors should be evaluated in this disease and approaches combining molecular targeted therapy and checkpoint inhibition could be of additional benefit. An extensive immune monitoring should be included in these studies.