Biliary tract cancers are aggressive tumors of which the incidence seems to increase. Resection with cancer-free margins is crucial for curative therapy. However, how often biliary intraepithelial neoplasia (BilIN) occurs in resection margins and what its clinical and therapeutic implications might be is largely unknown. We reexamined margins of resection specimens of adenocarcinoma of the biliary tree including the gallbladder for the presence of BilIN. When present, it was graded. The findings were correlated with clinicopathological parameters and overall survival. Complete examination of the resection margin could be performed on 55 of 78 specimens (71%). BilIN was detected in the margin in 29 specimens (53%) and was mainly low-grade (BilIN-1; N = 14 of 29; 48%). In resection specimens of extrahepatic cholangiocarcinoma, BilIN was most frequent (N = 6 of 8; 75%). BilIN was found in the resection margin more frequently in extrahepatic cholangiocarcinomas (P = 0.007) and in large primary tumors (P = 0.001) with lymphovascular (P = 0.006) and perineural invasion (P = 0.049). Patients with cancer in the resection margin (R1) had a significantly shorter overall survival than those with resection margins free of tumor (R0) irrespective of the presence of BilIN (R0 vs R1; P < 0.001) or BilIN grade (BilIN-positive vs BilIN-negative, P = 0.6, and BilIN-1 + 2 vs BilIN-3, P = 0.58). BilIN is frequently found in the surgical margin of resection specimens of adenocarcinoma of the biliary tract. Hepatopancreatobiliary surgeons will be confronted with this recently defined entity when an intraoperative frozen section of a resection margin is requested. However, this diagnosis does not require additional resection and in the intraoperative evaluation of resection, the emphasis should remain on the detection of residual invasive tumor.