Background: The FDA has approved immune checkpoint inhibitors for dMMR/MSI-H patients with solid tumors, but the efficacy varies greatly, so that more molecular biomarkers are needed to guide the clinical practice. Methods: In this study, the gene status of tumor tissues of 43 patients with dMMR gastric cancer previously identified by immunohistochemistry (IHC) was measured using TSO500, and the gene profile was preliminarily determined. Results: The genetic landscape is different between these 43 dMMR gastric cancer and TCGA as well as 200 cases of ordinary gastric cancer in China. The top five mutational genes in patients with dMMR gastric cancer were LRP1B (65.12%), RNF43 (62.79%), TP53 (58.14%), ARID1A (58.14%), BRCA2 (44.19%). In TCGA, the top five mutational genes were TP53 (48.1%), MUC16 (41.8%), SYNE1(32.1%), ARIDIA (31.4%) and LRP1B (30.7%). In 200 cases of patients with ordinary gastric cancer in China, the top five mutational genes were MUC6 (86.6%), MUC16 (84.6%), MACF1 (68.7%), DST (66.2%) and ZFHX4 (65.7%). The frequently abnormal signaling pathways in patients with dMMR gastric cancer were RTK/RAS (84%), P53 (77%), PI3K (74%), Notch (74%), Wnt (70%), Myc (47%),TGF-β(40%) signaling pathway. In addition, it was found that 40% of patients with dMMR gastric cancer were microsatellite-stable (MSS). Among the four dMMR biomarkers, 91.11% MLH1 negative or 86.67% MSH6 negative patients with dMMR gastric cancer were microsatellite-instable(MSI)–H, while only 11.11% MSH2 negative or 6.67% MSH6 negative patients with dMMR were MSI-H. Conclusions: This study provided important novel insight into the biology of dMMR gastric cancer and identified clinically genetic features for the future improvement management of patients with dMMR gastric cancer. It also suggested that in clinical practice, IHC is only suitable for preliminary screening.