DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in / are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while / / alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing. Over 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for / mutations by Sanger sequencing. mutations were identified in 308 (49%) PCs, while alterations in were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of mutations and alterations in had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively). In contrast to Sanger sequencing, preoperative NGS of PCF for / mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of / / alterations is a useful preoperative marker for advanced neoplasia.