PurposeResection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasivein vivoimaging approaches for precise TDLN identification.ProceduresFor visualization of the lymphatic drainage into the TDLNs by non-invasivein vivooptical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol−1) and IRDye® 800CW polyethylene glycol (PEG; 25,000–60,000 g mol−1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasivein vivoPET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol−1]) in a similar manner. Forex vivocross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis.ResultsThe clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasivein vivodetection by optical imaging.Ex vivoPatent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereasex vivoIRDye® 800CW PEG completely failed. In contrast, functional non-invasivein vivo18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN.Ex vivobiodistribution and autoradiography confirmed ourin vivo18F-FDG PET/MRI results.ConclusionsWhen taken together, our results demonstrate the feasibility of18F-FDG-PET/MRI as a valid method for non-invasivein vivo, intraoperative, andex vivoidentification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and byex vivooptical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11307-022-01797-z.