Introduction: The current study aimed to compare cytology using SurePath® (SP)‐LBC and biliary tissue histology (BTH) for the diagnosis of biliary disease. Methods: Between January 2014 and December 2016, 57 patients underwent endoscopic retrograde cholangiopancreatography for the diagnosis of biliary disease. Biliary cytological samples were processed using SP‐LBC and subsequently BTH was performed. A final diagnosis was confirmed by surgery (23 malignant cases) and clinical follow‐up (34 benign and malignant cases): 18 extrahepatic cholangiocarcinoma; 17 intrahepatic/hilar cholangiocarcinoma (intra/H‐CC); eight other malignant disease; and 14 benign biliary disease. The diagnoses made using SP‐LBC and BTH were classified into four categories: (1) benign; (2) indeterminate; (3) suspicious for malignancy/malignant; and (4) inadequate. In addition, diagnostic accuracy was compared between SP‐LBC and BTH. Results: Although 23% (13/57) of BTH samples were classified as inadequate, all SP‐LBC cases were classified as adequate. Among 43 malignant cases, 11 normal, four indeterminate and 28 suspicious for malignancy/malignant were found using SP‐LBC (26%, 9% and 65%, respectively), in contrast to 10 inadequate, nine normal, 10 indeterminate and 14 suspicious for malignancy/malignant observed using BTH (23%, 21%, 23%, and 33%, respectively). The identification of malignant cells was strikingly different between SP‐LBC and BTH. Furthermore, limited to intra/H‐CC, accuracy was significantly higher using SP‐LBC than using BTH (P < .001). Conclusions: SP‐LBC of the biliary tract is a useful and reliable method for diagnosing biliary malignant disease and has an advantage over BTH for detecting malignant cells and accurately diagnosing intra/H‐CC. [ABSTRACT FROM AUTHOR]