An accurate method to assess malignant lymph nodes in the mesorectum is needed. Dual-energy CT scans simultaneously with 2 levels of energy and thereby provides information about tissue composition based on the known effective Z value of different tissues. Each point investigated is represented by a certain effective Z value, which allows for information on its composition. We wanted to standardize a method for dual-energy scanning of rectal specimens to evaluate the sensitivity and specificity of benign versus malignant lymph node differentiation. Histopathological evaluation of the nodes was our reference. This was a descriptive and prospective study. Seventeen rectal specimens were examined in 2 series. The first series was conducted with 3 specimens from patients who were not given perioperative contrast; 3 had iodine-based contrast and 3 had gadolinium-based contrast. We concluded that iodine was the contrast agent of choice and therefore included 8 more patients in a second series, given iodine-based contrast, for further analysis. Quantitative imaging data were collected from 197 individual lymph nodes from 17 specimens, from patients with rectal cancer. We measured accuracy of differentiating benign from malignant lymph nodes by investigating the following: 1) gadolinium, iodine, and water concentrations in lymph nodes; 2) dual-energy ratio; 3) dual-energy index; and 4) effective Z value. Optimal discriminations between benign and malignant lymph nodes were obtained using the following cutoff values: 1) effective Z at 7.58 (sensitivity, 100%; specificity, 90%; and accuracy, 93%), 2) dual-energy ratio at 1.0 × 10 (sensitivity, 96%; specificity, 87%; and accuracy, 90%), 3) dual-energy index at 0.03 (sensitivity, 97%; specificity, 88%; and accuracy, 91%), and 4) iodine concentration at 2.58 μg/mL (sensitivity, 86%; specificity, 92%; and accuracy, 89%). The investigation is conducted on isolated surgical specimens from rectal cancer operations. Dual-energy CT can be performed on rectal specimens. The discrimination between benign and malignant nodes seems promising when using iodine as contrast.