Introduction Accurate assessment of the lymph node (LN) status is crucial in resectable perihilar cholangiocarcinoma (pCCA) to prevent major surgery in patients with extraregional metastatic LN (MLN). This study investigates the added value of preoperative Endoscopic Ultrasound (EUS) with or without Tissue Acquisition (TA) for the detection of MLN in patients with resectable pCCA. Methods In this retrospective, multicenter cohort study, patients with potentially resectable pCCA who underwent EUS preoperatively between 2010-2020, were included. The clinical impact of EUS-TA was defined as the percentage of patients who did not undergo surgical resection due to MLN found with EUS-TA. Findings of cross-sectional imaging were compared with EUS-TA findings and surgery. Results EUS was performed in 141 patients, of whom 107 (76%) had suspicious LN on cross-sectional imaging. Surgical exploration was prevented in 20 (14%) patients because EUS-TA detected MLN, of which 17 (85%) were extraregional. Finally, 74 (52%) patients underwent surgical exploration followed by complete resection in 40 (28%). MLN were identified at definitive pathology in 24 (33%) patients, of which 9 (38%) were extraregional and 15 (63%) regional. Discussion EUS-TA has the promise to be of value in patients with potentially resectable pCCA based on preoperative cross-sectional imaging, regardless of lymphadenopathy at cross-sectional imaging. A prospective study in which a comprehensive EUS investigation with LN assessment and EUS-TA of LN is performed routinely should confirm this promise.