Purpose <p>Recently, novel human epidermal growth factor receptor 2 antibody-drug conjugate therapies have shown remarkable efficacy in treating urothelial carcinoma, highlighting the growing need for comprehensive assessment of HER2 expression in tumors to identify patients who could benefit. However, non-invasive methods for assessing HER2 status in patients with urothelial carcinoma remain limited. This study aimed to preliminarily evaluate the clinical potential of [68Ga]Ga-HER2 affibody PET/CT for non-invasive assessment of HER2 expression in patients with urothelial carcinoma.</p> Materials and methods <p>A cohort of 25 patients with urothelial carcinoma was retrospectively included from a prospective parent study (NCT05535621), and sequential PET/CT using [68Ga]Ga-HER2 affibody and [18&#xa0;F] FDG was performed. HER2 status was assessed through pathological evaluation using immunohistochemistry, with scoring categories of 0, 1+, 2+, and 3+. Lesion uptake on PET/CT was quantified using the maximum standardized uptake value (SUVmax). Comparisons of SUVmax between primary tumor groups with different HER2 expression levels (HER2-positive IHC 2+, 3 + vs. HER2-negative IHC 0, 1+, and HER2-expression IHC 1+, 2+, 3 + vs. HER2-zero IHC 0) were conducted using nonparametric statistical tests. Additionally, the correlation between tumor SUVmax and HER2 expression status was analyzed, and the HER2 PET metastasis-positive rate at the patient level in metastatic urothelial carcinoma was determined.</p> Results <p>Twenty-five patients (mean age, 65.6 years ± 11.1 [SD]); 18 men) with urothelial carcinoma were enrolled. HER2 status in primary tumor was confirmed IHC 0 in four cases, IHC 1 + in three cases, IHC 2 + in six cases, IHC 3 + in three cases, and unknown in seven cases with potential HER2 expression. One patient with ureterostomy recurrence had HER2 IHC 1+, while another case was confirmed IHC 2 + through biopsy of metastatic lesion. In 16 participants with urothelial carcinoma, the SUVmax of HER2-positive primary tumors (IHC 2 + and 3+, <i>n</i> = 9) were higher than those of HER2-negative tumors (IHC 0 and 1+, <i>n</i> = 7) (median SUVmax, 7.07 ± 0.69 vs. 4.48 ± 1.14, <i>p</i> = 0.042) on [68Ga]Ga-HER2 affibody PET/CT. Additionally, a similar difference was observed between the HER2-expression group (IHC 1+, 2+, 3+, <i>n</i> = 12) and the HER2-zero group (IHC 0, <i>n</i> = 4) (median SUVmax, 6.97 ± 0.70 vs. 2.85 ± 0.19, <i>p</i> = 0.001). Primary tumors in 13 of the 16 patients were semi-quantitatively measurable on FDG PET/CT imaging, with no significant difference in FDG SUVmax between the HER2-positive and HER2-negative group (7 HER2-positive vs. 6 HER2-negative; median SUVmax: 19.56 ± 5.02 vs. 19.10 ± 3.13, <i>p</i> = 0.731). Spearman correlation analysis revealed a positive correlation between HER2 PET SUVmax and HER2 expression levels in primary urothelial carcinoma (<i>R</i> = 0.727, <i>p</i> = 0.001), whereas no correlation was found between FDG SUVmax and HER2 expression levels (<i>p</i> &gt; 0.05). In 14 patients with metastatic urothelial carcinoma, HER2 affibody PET/CT identified HER2 PET-positive metastatic lesions with a 92.86% positivity rate (13/14).</p> Conclusion <p>[68Ga]Ga-HER2 affibody PET/CT is a valuable non-invasive method for evaluating HER2 expression in patients with urothelial carcinoma.</p> Trial registration <p>NCT05535621. Registered 18 December 2021.</p>