ObjectiveTo investigate the value of the novel SpyGlass peroral choledochoscopy for the diagnosis of indeterminate biliary strictures. MethodsData of 66 patients with biliary strictures assessed by the second generation SpyGlass were collected in the retrospective study from January 2018 to January 2019. The imaging characteristics and type of biliary strictures were observed. The sensitivity, specificity, positive predictive value and negative predictive value of SpyGlass were evaluated with the results of pathology and long-term follow-up results as the golden standard. ResultsA total of 20 benign biliary stricture and 46 malignant biliary stricture were confirmed by pathology, other imaging examinations and long-term follow-up results. The sensitivity, specificity, positive predictive value and negative predictive value of SpyGlass were 100.0% (46/46), 90.0% (18/20), 95.8% (46/48) and 100.0% (18/18), respectively. Vessels of irregular morphology or brittle texture was the most common manifestation of malignant strictures under SpyGlass observation. ConclusionThe novel SpyGlass system can evaluate imaging features of most benign and malignant biliary strictures with high sensitivity.