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Endoscopic ultrasound (EUS) may offer a diagnostic tool through the combination of imaging and guided fine-needle aspiration of pancreatic cysts. The purpose of this investigation was to determine the most accurate test for differentiating mucinous from nonmucinous cysts.The results of EUS imaging, cytology, and cyst fluid biochemical markers were prospectively collected and compared in a large single-center study (776 patients) using histology or malignant cytology as the final diagnostic standard in 198 patients.The mean cyst fluid carcinoembryonic antigen (CEA) was greater in mucinous cysts (4703.0 ng/mL) compared with nonmucinous cysts (25.8 ng/mL) (P = 0.008). When using the optimal cutoff value of 109.9 ng/mL, the CEA was more accurate (86%, receiver operating characteristic area = 0.928) than EUS imaging (48%) and cytology (58%) in predicting a mucinous cyst (P < 0.0001). Malignant cysts had a mean cyst fluid CEA value (2558.2 ng/mL) similar to benign cysts (4700.2 ng/mL). Cytology (75%) more accurately diagnosed malignant cysts than EUS (66%) and CEA (62%) (P < 0.05).Cyst fluid CEA concentration provides a highly accurate test for the diagnosis of a mucinous cyst, but does not distinguish benign from malignant cysts. Cytology is the most accurate test for the diagnosis of a malignant cyst.