BACKGROUND: The chicken chorioallantoic membrane (CAM) model is a potential alternative to the mouse model based on the 3R principles. However, its value for determination of the in vivo behaviors of radiolabeled peptides through positron emission tomography (PET) imaging needed investigation. Herein, the chicken CAM tumor models were established, and their feasibility was evaluated for evaluating the imaging properties of radiolabeled peptides using a METHODS: Two human breast cancer cell lines were inoculated into chicken CAM and mice, respectively. The tumor-targeting potential and pharmacokinetic profile of a RESULTS: The tumor-formation time in chicken CAM model was shorter than that of mouse model. The uptake values of human epithelial growth factor receptor-2 (HER2)-positive Bcap37 tumors in chicken CAM and mouse models were 5.36 ± 0.26% ID/g and 5.26 ± 0.43% ID/g at 30 min postinjection of CONCLUSION: In this study, the CAM tumor model was successfully prepared. The chicken CAM model is a novel tool for quickly determining the in vivo properties of radiolabeled peptides targeting biomarkers. It may be beneficial for early monitoring of the therapeutic effect of a new drug through PET imaging with specific peptides.