BACKGROUND AND AIMS: Biopsy of the ampulla of Vater may be performed to evaluate for ampullary adenomas, suspected ampullary tumors and immunohistological staining for autoimmune pancreatitis. Ampullary biopsies are commonly performed at the time of endoscopic retrograde cholangiopancreatography (ERCP). Due to the well-established complication rate following ERCP, the contribution of ampullary biopsy as a potential independent risk factor would require a controlled comparison. METHODS: A matched-pairs, case-control analysis was performed for patients undergoing ERCP with or without ampullary biopsy. The analysis involved a retrospective review of adult patients at a tertiary-care center who underwent ampullary biopsies during ERCP compared (via procedural complexity) with a matched control group who underwent ERCP without ampullary biopsies. RESULTS: Of 159 procedures involving ampullary biopsy, 54 ERCPs that met the inclusion criteria were performed with ampullary biopsy and included in the analysis cohort. This cohort was compared with 54 patients undergoing ERCP without ampullary biopsy, matched by American Society for Gastrointestinal Endoscopy (ASGE) grade of procedural complexity. There were no patients with sphincter of Oddi dysfunction. Ampullary biopsies suggested a diagnosis in 75.9% of the procedures including 12 adenomas, 5 adenocarcinomas and 1 intraductal papillary mucinous neoplasm. Including major and minor complications, the overall complication rate with biopsy (9.3%) was equivalent to the complication rate in the control group without ampullary biopsy (9.3%, P>0.99). The incidence of post-procedure pancreatitis was not significantly different between the two groups (5.6% vs 3.7%, P=0.6). Age and pancreatic duct manipulation, but not ampullary biopsy, were associated with complications on multivariate analysis in the study population. CONCLUSIONS: Ampullary biopsy performed during ERCP had a high diagnostic yield and was not associated with an increased rate of post-procedure complications or pancreatitis when compared with ERCP alone.