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Background Peroral cholangioscopy (POCS)-guided forceps biopsy is a method for diagnosing indeterminate biliary strictures and for the preoperative identification of the exact perihilar and distal margins of biliary tract cancer (BTC). However, POCS-guided forceps biopsy may result in an insufficient amount of specimen at times. Therefore, we evaluated the adequate tissue acquisition rate and the factors affecting the adequate tissue acquisition of POCS-guided forceps biopsy for the biliary tract. Methods Patients who underwent POCS-guided forceps biopsy for biliary disease between September 2016 and October 2018 at our hospital were enrolled retrospectively. We evaluated the adequate tissue acquisition rate of POCS-guided forceps biopsy for the biliary lesion and that for non-stenotic bile duct. In addition, the factors affecting the adequate tissue acquisition rate of POCS-guided forceps biopsy were evaluated. Results We enrolled 47 patients with biliary disease and performed POCS-guided forceps biopsy for biliary lesion and POCS-guided forceps mapping biopsy for non-stenotic bile duct in 40 and 36 patients, respectively. The adequate tissue acquisition rates of POCS-guided forceps biopsy for biliary lesions and that for non-stenotic bile duct were 86.4%, and 68.9%, respectively. In the multivariate logistic regression analyses, age, and previous biliary stenting before POCS were factors affecting the adequate tissue acquisition rate of POCS-guided forceps biopsy for the biliary lesion. For non-stenotic bile duct, the location of the biliary lesion, endoscopic sphincterotomy (EST), and procedure time of POCS were factors affecting the adequate tissue acquisition rate of POCS-guided forceps mapping biopsy. Conclusions Previous biliary stenting was a factor affecting a low tissue acquisition rate of POCS-guided forceps biopsy for the biliary lesion. In the POCS-guided forceps mapping biopsy, the location of the biliary lesion, EST, and procedure time were factors affecting tissue acquisition rates.