ObjectivesEndoscopic ultrasound‐guided fine‐needle aspiration and fine‐needle biopsy (EUS‐FNA/FNB) is not fully established as a pathological sampling tool for gallbladder lesions due to limited evidence. We therefore aimed to clarify the effectiveness and safety of this procedure in a large‐population cohort.MethodsThis study retrospectively evaluated the diagnostic yield of EUS‐FNA/FNB for accurately differentiating between benign and malignant gallbladder lesions. Puncture targets included the gallbladder mass, lymph node, and liver mass. Adverse events and factors associated with diagnostic accuracy were analyzed as well.ResultsIn 187 patients with gallbladder lesions undergoing EUS‐FNA/FNB, 18 benign lesions and 169 malignant lesions were identified. Overall sampling adequacy was 98% (184/187). The diagnostic accuracy of EUS‐FNA/FNB was 97% (182/187), sensitivity was 97% (164/169), and specificity was 100% (18/18). A single postprocedural complication (minor bleeding) was recorded in one patient. In the 169 cases of malignancy, 203 sites were punctured for pathological sampling of the primary mass (n = 94), lymph node (n = 79), and metastatic liver mass (n = 30). No significant difference was found for diagnostic accuracy among the puncture sites (P = 0.70). In cases having specimens obtained from the primary mass, the accuracy of those targeting liver invasion sites was significantly higher than that of other sites (98% vs. 83%, P < 0.01).ConclusionEUS‐FNA/FNB demonstrated clinical usefulness and safety for the pathological diagnosis of gallbladder lesions, with high diagnostic yield and a low incidence of adverse events. Targeting the site of liver infiltration may improve the diagnostic rate of EUS‐FNA/FNB in the primary mass.