Abstract Background Preoperative biopsy of pancreatic lesions suspected of malignancy is controversial. Methods A systematic Medline literature search was carried out. Diagnostic studies reporting quantitative preoperative pancreatic biopsy data were evaluated. Results The analysis included 53 studies, mostly of a retrospective nature. Despite acceptable rates for sensitivity and specificity, the negative predictive value of percutaneous and endoscopic ultrasonography-guided biopsies was 60–70 per cent. Biopsy results were considered to be essential for directing non-surgical therapy in advanced disease. However, they were of limited value in planning the treatment of resectable solid or cystic tumours, or focal lesions in the setting of chronic pancreatitis. Conclusions Biopsy of suspected pancreatic malignancies with systemic spread or local irresectability is indicated for planning palliative or neoadjuvant therapy. Preoperative biopsy of potentially resectable pancreatic tumours is not generally advisable, as malignancy cannot be ruled out with adequate reliability.