Noninvasive characterization of lymph node involvement in cancer is an enduring onerous challenge. In rectal cancer, pathologic lymph node status constitutes the most important determinant of local recurrence and overall survival, and patients with involved lymph nodes may benefit from preoperative chemo and/or radiotherapy. However, knowledge of lymph node status before surgery is currently hampered by limited imaging accuracy. Here, we introduce Susceptibility-Perturbation MRI (SPI) as a novel source of contrast to map malignant infiltration into mesorectal lymph nodes. SPI involves multigradient echo (MGE) signal decays presenting a nonmonoexponential nature, which we show is sensitive to the underlying microstructure via susceptibility perturbations. Using numerical simulations, we predicted that the large cell morphology and the high cellularity of tumor within affected mesorectal lymph nodes would induce signature SPI decays. We validated this prediction in mesorectal lymph nodes excised from total mesorectal excision specimens of patients with rectal cancer using ultrahigh field (16.4 T) MRI. SPI signals distinguished benign from malignant nodal tissue, both qualitatively and quantitatively, and our histologic analyses confirmed cellularity and cell size were the likely underlying sources for the differences observed. SPI was then adapted to a clinical 1.5 T scanner, added to patients' staging protocol, and compared with conventional assessment by two expert radiologists. Nonmonoexponential decays, similar to those observed in the ex vivo study, were demonstrated, and SPI classified lymph nodes more accurately than standard high-resolution T2-weighted imaging assessment. These findings suggest this simple, yet highly informative, method can improve rectal cancer patient selection for neoadjuvant therapy. SIGNIFICANCE: These findings introduce an MRI methodology tailored to detect magnetic susceptibility perturbations induced by subtle alterations in tissue microstructure.