Objective Biliary brush cytology is an important diagnostic tool in the evaluation of pancreatobiliary malignancies. However, it is difficult to distinguish between malignant and benign cells. The present study evaluated the utility of immunocytochemical expression of Claudin‐18 and Maspin in brushing cytology specimens of pancreatobiliary lesions in the diagnosis of pancreatobiliary malignancies. Methods The study retrospectively assessed biliary and pancreatic duct brushing cytology specimens of 43 patients whose pancreatobiliary lesions were histologically diagnosed at the University of Miyazaki Hospital. Scanty cellularity slides and cases with no histological confirmation were excluded. Alcohol‐fixed and Papanicolaou‐stained slides were immunostained with monoclonal antibodies to Claudin‐18 and Maspin. Results Of the 43 patients, 35 (81.4%) were finally histologically diagnosed with invasive adenocarcinomas. The sensitivity of routine cytology for the detection of malignancy was 63%, and the specificity was 100%. The sensitivity of cytology in combination with immunocytochemical expression of Claudin‐18 (89%) or Claudin‐18 and/or Maspin (97%) was significantly higher than that of cytology alone (P < 0.01). Conclusion Immunocytochemical staining for Claudin‐18 and Maspin improved the diagnostic sensitivity for pancreatobiliary adenocarcinomas. Authors claim that this is the first study examining the Immunocytochemical staining for Claudin‐18 and Maspin in biliary brushing cytology specimens. The result of this retrospective study in 43 patients showed that the addition of these two immunological stains can improve the diagnostic sensitivity of the pancreaticobiliary adenocarcinomas.