Objective: The current molecular classification system for gastric cancer covers genomic, molecular, and morphological characteristics. Non-etheless, classification of gastric cancer based upon DNA damage repair is still lacking. Here, we defined DNA damage repair-based subtypes across gastric cancer and identified clinicopathological, tumor microenvironment and pharmacogenomic features. Methods: Unsupervised clustering analysis was executed in the TCGA-STAD cohort based upon the transcriptional expression profiling of DNA damage repair genes. LASSO computational approach was adopted for generating a DNA damage repair-relevant gene signature. The identified subtypes or signature were externally verified in the GSE84426 or GSE84433 cohort. The transcriptional levels of immunomodulators, abundance of immune cells and somatic mutations were measured, respectively. Immunotherapeutic response, and drug sensitivity were investigated. The DNA damage repair-relevant genes were further experimentally verified. Results: Two DNA damage repair-based subtypes were identified, with the notable heterogeneity in prognostic stratification, tumor microenvironment and somatic mutations. The gene signature was generated for risk stratification and prognostic prediction, which was in relation to immunomodulators and immune cells. High-risk cases were more likely to respond to immunotherapy, with distinct pharmacogenomic landscapes between low- and high-risk groups. Higher levels of PAPPA2, MPO, MAGEA11, DEPP1, CPZ, and COLEC12 and lower level of CYTL1 were proven in gastric cancer cells versus controls. Silencing CYTL1 facilitated intracellular ROS accumulation and suppressed migration in gastric cancer cells. Conclusion: Collectively, the DNA damage repair-based classification is a suitable complement to existing molecular classification system, and the quantitative gene signature provides a robust tool in selecting specific therapeutic options.Copyright © 2023 Kong, Wang and Wang.