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Biliary strictures that are suspicious for cholangiocarcinoma (CCA) are commonly encountered in clinical practice in patients with and without primary sclerosing cholangitis (PSC). A definitive histologic diagnosis of CCA via endoscopic retrograde cholangiography (ERCP) is often not obtainable with standard biliary brush cytology. Peroral cholangioscopy is an additional tool to help provide a diagnosis of CCA in patients with suspicious biliary strictures. Aim of the study was to assess the use of peroral cholangioscopy in patients with and without PSC and indeterminate biliary strictures.Retrospective study.25 patients were included in the study. All patients underwent ERCP with peroral cholangioscopy. Tissue samples obtained included routine cytology, fluorescent in-situ hybridization, and cholangioscopic-directed forceps biopsies. The operating characteristics of cholangioscopy to detect malignancy in 18 PSC patients with suspected cholangiocarcinoma were a sensitivity of 75%, specificity of 55%, and a positive predictive value (PPV) of 23%, and a negative predictive value of 92%. In 7 non-PSC patients with suspected cholangiocarcinoma these values sensitivity=100%, 25%, 50%, and NPV=100%, respectively. The overall operating characteristics of cholangioscopy to detect malignancy in all 25 patients with suspected cholangiocarcinoma were: sensitivity of 86%, specificity of 50%, PPV of 32%, and NPV of 93%.Cholangioscopy helps identify sites for tissue acquisition in PSC and non-PSC patients with biliary strictures suspicious for malignancy.