Background: Prognosis of patients with gastric cancer (GC) with peritoneal metastasis (PM) still remains poor, and new treatment strategies are needed. Claudins (CLDNs) are main components of tight junctions that physiologically mediate cell–cell adhesion and regulate selective permeability in epithelial cellular sheets. Recent clinical trials have demonstrated that monoclonal antibodies (mAb) to CLDN18.2 have been shown to improve the outcome of the patients with unresectable metastatic GC, especially with undifferentiated histology. However, the clinical effects of the anti-CLDN18.2 mAb for patients with PM is unclear. In this study, we evaluated the expression of CLDN18.2 in primary tumor and metastatic nodules in peritoneum of GC using immunohistochemical (IHC) staining. Methods: In 42 patients diagnosed with stage IV GC with PM at our Department from 2014 to 2022, biopsy samples of primary tumor and peritoneal nodules were obtained and the expression of CLDN18.2 in tumor cells by IHC staining using specific antibody for CLDN18.2 (clone: 43-14A, prediluted; Ventana) followed by the incubation with secondary antibody and the primary antibody binding visualized using the DAB IHC Detection Kit. The intensity of the membrane and cytosolic staining was classified as 0 (no reactivity), 1+ (weak), 2+ (moderate), and 3+ (strong). Samples were defined as CLDN18.2-positive when tumor cells showed specific staining with at least 1+ intensity in tumor cells. Percentage of overall CLDN18.2 positive cells was determined by the estimated number of CLDN18.2+ cells divided by the estimated overall number of tumor cells in any fractions. Her2 expression was also examined in parallel. Results: The histology type of primary tumor was differentiated in 7, undifferentiated in 33, and unknown in 2 patients. CLDN18.2-positivity was detected in 31 (74%) of primary tumors, including 5 (71%) differentiated and 26(79%) undifferentiated tumors. CLDN18.2 was positively detected in 15 (35%) disseminated nodules, in 4 (57%) differentiated and 11 (33%) in undifferentiated cases, respectively. All these cases also showed CLDN18.2-positve in primary sites. However, expression of CLDN18.2 in PM was not detected in 16 (52%) cases which showed CLDN18.2-positive for primary tumor. Moderate-to-strong CLDN18.2 expression (≥2+ membrane staining intensity in ≥40% of tumor cells) was observed in 13 (31%) of primary tumor, but only in 2 (5%) of PM. HER2 was positive in 5 (12%) patients, and no correlation was observed between HER2 and CLDN18.2 expression. Conclusions: CLDN18.2 expression did not show significant difference depending on histology. Although the expression of CLDN18.2 in PM showed homology with that in biopsy sample of primary gastric tumor, its expression level generally decreased in PM. Investigation of CLDN18.2 expression in peritoneal nodules might be necessary to predict the clinical response of CLDN18.2 mAb for GC with PM.