Although 18F-fluorodeoxyglucose (18F-FDG) uptake can be used for the non-invasive detection and monitoring of allograft rejection by activated leucocytes, this non-specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a 131I-radiolabelled anti-Toll-like receptor 5 (TLR5) mAb for non-invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non-specific radiotracer 18F-FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo-treated group or allo-rejection group), were injected with 131I-anti-TLR5 mAb, 18F-FDG, or mouse isotype 131I-IgG, respectively. Whole-body phosphor-autoradiography and ex vivo biodistribution studies were obtained. Whole-body phosphor-autoradiography showed 131I-anti-TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The 131I-anti-TLR5 mAb had significantly higher graft uptake and target-to-non-target ratio in the allo-treated group, as determined by semi-quantification of phosphor-autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high 18F-FDG uptake was not observed in the allo-treated group. The highest allograft-skin-to-native-skin ratio (A:N) of 131I-anti-TLR5 mAb uptake was significantly higher than the ratio for 18F-FDG (7.68 versus 1.16, respectively). 131I-anti-TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of 131I-IgG was comparable in both groups. We conclude that radiolabelled anti-TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.