Negative circumferential resection margins (CRM) are the cornerstone for curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients tumor-positive resection margins are detected upon histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool to evaluate the CRM directly after surgical resection in order to improve tumor-negative CRM rates. Methods: LARC patients that were treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A (VEGFA), 2-3 days before surgery (NCT01972373). To evaluate the CRM status, back-table fluorescence-guided imaging (FGI) was performed of the fresh surgical resection specimens (N = 8). These results were correlated to histopathology. Secondly, to determine the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity (MFI) cut-off value was determined on the formalin-fixed tissue slices (N = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in one of two patients (50%) with a tumor-positive CRM. The other patient showed low fluorescence intensities, although (sub-)millimeter tumor deposits were present <1 mm of the CRM. FGI correctly identified 5/6 tumor-negative CRMs (83%). The one patient with false-positive findings had a marginal negative CRM of only 1.4 mm. ROC analysis of fluorescence intensities of formalin-fixed tissue slices gave an optimal MFI cut-off value for tumor detection of 5,775 (sensitivity and specificity of 96.16% and 80.39% respectively). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 ± 2.5 (mean ± SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI to evaluate the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision-making with regard to extending resections or applying intraoperative radiation therapy in case of positive CRMs. Copyright © 2019 by the Society of Nuclear Medicine and Molecular Imaging, Inc.