It is difficult to distinguish pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis (CP) when stricture is present in the pancreatic duct. Endoscopic brushing cytology is a convenient method for investigating strictures in the pancreatic duct, however, the diagnostic sensitivity of this method for PDAC is reported to be low (40–70%). Recently, we revealed that MSX2 is frequently expressed in PDAC cells but not in normal cultured pancreatic duct or stellate cells. Thus, we analyzed MSX2 expression levels in brushing samples to examine whether this would differentiate PDAC from CP. Cytologic brushing specimens were obtained from pancreatic duct strictures during endoscopic retrograde cholangiopancreaticography in 82 patients. The brushing fluid was subjected to cytological diagnosis and RNA extraction. The expression level of MSX2 was evaluated by one-step real-time RT-PCR. MSX2 expression levels were significantly higher in PDAC than in CP (P = 0.0000007), and the expression level was associated with positive cytology (P = 0.013). The sensitivity, specificity, and diagnostic accuracy for PDAC of cytology and MSX2 expression in ductal strictures were: 47.4%, 100%, and 63.4%, and 73.7%, 84.0%, and 79.3%, respectively. The sensitivity and accuracy of MSX2 expression levels for diagnosis were much higher than those of cytology. This suggests that the evaluation of MSX2 levels in endoscopic retrograde cholangiopancreaticography brushing samples would be useful for distinguishing PDAC from CP. (Cancer Sci 2011; 102: 157–161)