Background: Peritoneum is a common site of recurrence and metastasis of gastric cancer. The traditional clinical methods have poor sensitivity for the diagnosis of peritoneal metastasis, and there is no effective marker for predicting peritoneal metastasis of gastric cancer. In this study, the genetic profiles of primary tumors in gastric cancer patients with and without peritoneal metastasis, as well as the genetic alterations before and after metastasis in the same patient were analyzed to explore the genetic alterations associated with peritoneal metastasis. Methods: 232 patients with stage Ⅳ gastric cancer were divided into PM group and non-PM group according to whether there was peritoneal metastasis, DNA based Next Generation Sequencing (NGS) with a 1021 gene panel was performed on their samples. Clinical information was obtained synchronously from physicians and surgeons. Results: There was no significant difference in baseline clinical characteristics between patients in PM and non-PM groups. Molecular analysis revealed that in the PM group, the incidence of CDH1, CDH23, HDAC1, IDH1, KRAS, MED12, RHOA, NOTCH2, TGFBR1 mutations was significantly higher in PM group than that in non-PM group, and these mutated genes were mostly located in wnt/β- catenin and MAPK signaling pathway. On the contrary, the incidence of ERBB2 and CCNE1 amplification was significantly higher in non-PM group than in the other group. Further analysis of samples before and after peritoneal metastasis of 13 patients did not find significant differences in genetic alterations. Conclusions: Compared with other sites, the primary tumor of gastric cancer patients with peritoneal metastasis seems to have its unique molecular characteristics, and large-panel NGS can help us identify this part of patients. Future research may need to clarify whether more radical treatment strategies are needed for these patients.