Background: One of the controversial issues in the diagnosis of pancreatic neuroendocrine tumours (pNETs) is the accurate prediction of their clinical behaviour. Objectives: The aim of the study was to evaluate the role of endoscopic ultrasound (EUS) biopsy in the diagnosis and grading of pNETs in a certified ENETS Center. Methods: A prospectively maintained database of EUS biopsy procedures was retrospectively reviewed to identify all consecutive patients referred to a certified ENETS Center with a suspicion of pNET between June 2014 and April 2017. The cytological and/or histological specimens were stained and the Ki-67 labeling index was evaluated. In patients undergoing surgery, the grade obtained with EUS-guided biopsy was compared with the final histological grade. The grade was evaluated according to the 2017 WHO classifications and grading. Results: The study population included 59 patients. EUS biopsy material reached an adequacy of 98.3% and was adequate for Ki-67 evaluation in 84.7% of cases. Twenty-nine patients (49.2%) underwent surgery. Of these, 25 patients had Ki-67 evaluated on EUS biopsy: the agreement between EUS biopsy grading and surgical specimen grading was 84%. Conclusion: EUS biopsy is an accurate method for the diagnosis and grading of pNETs based on the WHO 2017 Ki-67 labelling scheme.