Purpose: Peritoneal lavage cytology is part of the routine staging workup for patients with advanced gastric cancer. However, no quality assurance study has been conducted to show variations or biases in peritoneal lavage cytology results. The aim of this study was to demonstrate a test execution variation in peritoneal lavage cytology between investigating surgeons. Materials and Methods: A prospective cohort study was designed for determination of the positive rate of peritoneal lavage cytology using a liquid-based preparation method in patients with potentially curable advanced gastric cancer (cT2~4/N0~2/M0). One hundred thirty patients were enrolled and underwent laparotomy, peritoneal lavage cytology, and standard gastrectomy, which were performed by 3 investigating surgeons. Data were analyzed using the chi-square test and a logistic regression model. Results: The overall positive peritoneal cytology rate was 10.0%. Subgroup positive rates were 5.3% in pT1 cancer, 2.0% in pT2/3 cancer, 11.1% in pT4a cancer, and 71.4% in pT4b cancer. In univariate analysis, positive peritoneal cytology showed significant correlation with pT stage, lymphatic invasion, vascular invasion, ascites, and the investigating surgeon. We found the positive rate to be 2.1% for surgeon A, 10.2% for surgeon B, and 20.6% for surgeon C (P=0.024). Multivariate analysis identified pT stage, ascites, and the investigating surgeon to be significant risk factors for positive peritoneal cytology. Conclusions: The peritoneal lavage cytology results were significantly affected by the investigating surgeon, providing strong evidence of test execution variation that could be related to poor diagnostic accuracy and stage migration in patients with advanced gastric cancer.