Pancreatic ductal adenocarcinoma (PDA) is rarely curable due to regional/metastatic spread at diagnosis. Identification of molecular markers may enhance diagnosis and early detection of PDA. The 2-electron reductase, NAD(P)H:quinone oxidoreductase (NQO1) has been found to be overexpressed in many solid tumors including PDA, and may be a useful clinically relevant diagnostic marker of malignancy. For this study, we used 37 surgical resection cases: 24 PDAs and 13 benign pancreatic tissue specimens. An additional 16 specimens from pancreatic endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) were included as a pilot series. NQO1 was detected by avidin-biotin based immunohistochemical and immunocytochemical methods. Both staining intensity and proportion of NQO1 positive tumor cells were scored. Moderate to strong (2 to 3+) staining for NQO1 was detected in 22/24 (92%) surgically resected PDAs, 9/9 (100%) EUS-FNAs with malignant diagnoses, one cytologically atypical but not diagnostic for malignancy EUS-FNA, and 1/6 (17%) EUS-FNAs initially diagnosed as negative for malignancy. Subsequent histologic assessment confirmed malignancy in all 9 cytologically positive EUS-FNAs and in the atypical case. The NQO1 positive case initially diagnosed as negative for malignancy showed no evidence of carcinoma on subsequent tissue biopsy. NQO1 staining was also observed in some benign ducts/cells; however, correlation of NQO1 expression with cellular morphology assessment minimizes the risk of false positive diagnosis. NQO1 is consistently overexpressed in PDA. Although NQO1 is observed in some benign tissue components, this marker may be a clinically useful diagnostic adjunct for detection of PDA, independent of tumor grade/stage.