Purpose: The role of peritoneal washing cytology in determining further treatment strategies after surgery for gastric cancer remains unclear. One reason for this is the fact that optimal procedures to increase the accuracy of predicting peritoneal metastasis have not been established. The aim of this study was to evaluate the efficacy of cytology using samples harvested from two different abdominal cavity sites during gastric cancer surgery. Materials and Methods: We prospectively recruited 108 patients who were clinically diagnosed with locally advanced gastric cancer (higher than cT1 stage disease). Peritoneal washing fluids were collected from the pouch of Douglas and the subphrenic area. Patients were prospectively followed up for 2 years to determine the recurrence and survival rates. Results: Thirty-three patients dropped out of the study for various reasons, so 75 patients were included in the final analysis. Seven patients (9.3%) showed positive cytology findings, of whom, three showed peritoneal recurrence. Tumor size was the only factor associated with positive cytology findings (P=0.037). The accuracy and specificity of cytology for predicting peritoneal recurrence were 90.1% and 94.2%, respectively, whereas the sensitivity was 50.0%. The survival rate did not differ between patients with positive cytology findings and those with negative cytology findings (P=0.081). Conclusions: Peritoneal washing cytology using samples harvested from two different sites in the abdominal cavity was not able to predict peritoneal recurrence or survival in gastric cancer patients. Further studies will be required to determine whether peritoneal washing cytology during gastric cancer surgery is a meaningful procedure.