Detection and removal sentinel lymph nodes (SLN) is important in the diagnosis and treatment of breast cancer and malignant melanoma. The SLN is the first regional lymph node draining the cancer tumor and if the cancer has spread it is most likely to find cancer cells in the SLN. In this study we have been able to detect multimodal superparamagnetic iron oxide nanoparticles (SPIO-NP) in rat SLNs in situ using magnetomotive ultrasound imaging (MMUS). In MMUS a time-varying external magnetic field acts to move the NPs and, thus, the NP-laden tissue. This movement can be detected by proper processing of ultrasound data. We have recently developed an MMUS algorithm, based on quadrature detection and phase gating at the frequency of NP displacement, and this is the first study where the algorithm is evaluated in animals. For both higher NP-concentration, as well as smaller NPs, we found that the MMUS data showed a larger displacement (1.56 ± 0.43 and 1.94 ± 0.54 times larger, respectively). The MMUS displacement also increased with a lower excitation frequency (1.95 ± 0.64 times larger for 5 Hz compared to 15 Hz) and higher excitation voltage (2.95 ± 1.44 times larger for 30V compared to 10V). The results from this study show that the MMUS technique has potential to be used as bedside guidance during SLN surgery, well as being used as standalone technique in a number of other applications such as stem cell tracking and cardiovascular research.