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This study assessed the ability of spectral analysis of endoscopic ultrasound (EUS) RF signals acquired in humans in vivo to distinguish between (1) benign and malignant intraabdominal and mediastinal lymph nodes and (2) pancreatic cancer, chronic pancreatitis, and normal pancreas. Mean midband fit, slope, intercept, and correlation coefficient from a linear regression of the calibrated RF power spectra were computed over regions of interest defined by the endoscopist. Linear discriminant analysis was then performed to develop a classification of the resulting spectral parameters. For lymph nodes, classification based on the midband fit and intercept provided 67% sensitivity, 82% specificity, and 73% accuracy for malignant vs. benign nodes. For pancreas, classification based on midband fit and correlation coefficient provided 95% sensitivity, 93% specificity, and 93% accuracy for diseased vs. normal pancreas and 85% sensitivity, 71% specificity, and 85% accuracy for pancreatic cancer vs. chronic pancreatitis. These promising results suggest that mean spectral parameters can provide a non-invasive method to quantitatively characterize pancreatic cancer and lymph malignancy in vivo.