High-frequency quantitative ultrasound (QUS) permits characterization of tissue microstructure using system-independent estimates. In this study, freshly-excised lymph nodes from cancer patients were evaluated using specifically designed three-dimensional (3D) QUS methods. The long-term objective was to develop 3D QUS methods for detecting metastases. Detection of metastases is critically important for cancer staging and treatment planning. A custom laboratory scanning system was used to acquire radio-frequency (RF) data in 3D from excised lymph nodes using a 26-MHz center-frequency transducer. Overlapping 1-mm cylindrical regions-of-interest (ROIs) of the RF data were processed to yield 13 QUS estimates associated with tissue microstructure. QUS estimates were obtained from more than 250 nodes from more than 150 colorectal-, gastric-, and breast-cancer patients. Cancer-detection performance was assessed for individual estimates and linear combinations of estimates. ROC results demonstrated excellent classification. For colorectal- and gastric-cancer nodes, the areas under the ROC curves (AUCs) were above 0.94. Slightly poorer results (AUC 0.87) were obtained for breast nodes. Images based on QUS parameters also permitted localization of cancer foci in some micrometastatic cases. Therefore, these advanced 3D QUS methods potentially can be valuable for detecting small metastatic foci in dissected lymph nodes.