Background: Anti-programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra-and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy. This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules. Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4N+ M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+) T-cell density in primary tumors and PD-1 expression on CD8(+) T cells were detected with immunofluorescence. Univariate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival. Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors (45.4% vs. 38.7%, P = 0.005); the positive rate of PD-1 on CD8(+) T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes (both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression, PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis. Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stage T1-4N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.