Background: It remains unclear whether the 22- and 25-gauge needles have equal diagnostic yields in endoscopic ultrasonography–fine needle aspiration of pancreatic masses. Methods: Patients presenting with pancreatic solid masses were prospectively included between July 2010 and June 2012. Each patient underwent ultrasonography–fine needle aspiration using two different needle sizes (22- and 25-gauge needles) during the same endoscopic session, the 25 and 22 needle sequence being randomized. Cytological preparations included smear cytology, ThinPrep and Cell Block. Specimens were analyzed for diagnosis, cellularity, amount of blood and digestive contamination. Final diagnosis was reached by the final pathological report, clinical and radiological follow-up. Results: Thirty-seven patients were included among them 34 subjects had malignant pancreatic neoplasm on final diagnosis. Pancreatic masses were located in the head of pancreas in 73% of cases. The mean size of the lesions was 34.6 ± 14.4 mm. Diagnostic yields of the 25- and 22-gauge needles were 85.3% (95%CI: 72.6-98) and 88.2% (95%CI:76.7-99.7) respectively (p > 0.05). Diagnosis was obtained with smear cytology alone in 58.8% patients with the 22-gauge needle and in 55.9% patients with the 25-gauge needle, in 64.5% patients with Cell Block for both needles and in 76.5% with ThinPrep for both needles. The quality of specimens was comparable with the two needles. No complication occurred. Conclusion: The 25- and 22-gauge needles carry the same diagnostic yield when performing ultrasonography– fine needle aspiration of solid pancreatic masses. ISRCTN69545565 Controlled trials.