Purpose <p>Human epidermal growth factor receptor 2 (HER2)-targeted antibody-drug conjugates (ADCs) in breast cancer is progressing rapidly, asking for precise categorization of HER2 expression. Our aim was to explore the HER2-affibody uptake characteristics in breast cancer and to find potential biomarkers for differentiating HER2 status, in comparison with ¹⁸F-FDG PET/CT.</p> Methods <p>In this prospective study conducted from May 2024 to Jan 2025, participants with newly diagnosed breast cancer underwent both ⁶⁸Ga-HER2-affibody and ¹⁸F-FDG PET/CT examinations. The relationship between PET parameters (such as maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), tumor-to-background SUV ratio (TBR)) and HER2 expression statuses (HER2-zero, -low and -positive) was analyzed.</p> Results <p>A total of 57 female participants (58 primary lesions) with newly diagnosed breast cancer were included. In the paired comparison of ⁶⁸Ga-HER2-affibody and ¹⁸F-FDG PET/CT, HER2-TBR was higher than FDG-TBR in the HER2-low and HER2-positive group, respectively (<i>P</i> &lt; 0.001). HER2-SUVmax and HER2-SUVmean exhibited differences across most comparisons, especially HER2-zero vs. -low (<i>P</i> &lt; 0.05). HER2-SUVmean emerged as an independent predictor (<i>P</i> = 0.01; OR: 2.8) for differentiating HER2-low/positive and -zero, yielding an AUC of 0.91. Under detailed HER2 immunohistochemistry, HER2-SUVmax, -SUVmean and -TBR demonstrated intergroup differences (<i>P</i> &lt; 0.05).</p> Conclusion <p>HER2-targeted PET/CT imaging demonstrated distinct advantages over ¹⁸F-FDG PET/CT imaging in characterizing the HER2 status of primary lesions. Parameters based on HER2-affibody differed significantly among different HER2 statuses, and might be potential imaging indicators for screening patients with HER2-low/positive who could benefit from HER2-targeted ADCs.</p> ClinicalTrials.gov identifier <p>NCT06289517 (2024-03-04).</p>