Imaging using affibody molecules enables discrimination between breast cancer metastases with high and low expression of HER2, making appropriate therapy selection possible. This study aimed to evaluate if the longer half-life of ⁶⁴ Cu ( T1/2 = 12.7 h) would make ⁶⁴ Cu a superior nuclide compared to ⁶⁸ Ga for PET imaging of HER2 expression using affibody molecules. The synthetic ZHER2:S1 affibody molecule was conjugated with the chelators NOTA or NODAGA and labeled with ⁶⁴ Cu. The tumor-targeting properties of ⁶⁴ Cu-NOTA-ZHER2:S1 and ⁶⁴ Cu-NODAGA-ZHER2:S1 were evaluated and compared with the targeting properties of ⁶⁸ Ga-NODAGA-ZHER2:S1 in mice. Both ⁶⁴ Cu-NOTA-ZHER2:S1 and ⁶⁴ Cu-NODAGA-ZHER2:S1 demonstrated specific targeting of HER2-expressing xenografts. At 2 h after injection of ⁶⁴ Cu-NOTA-ZHER2:S1, ⁶⁴ Cu-NODAGA-ZHER2:S1, and ⁶⁸ Ga-NODAGA-ZHER2:S1, tumor uptakes did not differ significantly. Renal uptake of ⁶⁴ Cu-labeled conjugates was dramatically reduced at 6 and 24 h after injection. Notably, radioactivity uptake concomitantly increased in blood, lung, liver, spleen, and intestines, which resulted in decreased tumor-to-organ ratios compared to 2 h postinjection. Organ uptake was lower for ⁶⁴ Cu-NODAGA-ZHER2:S1. The most probable explanation for this biodistribution pattern was the release and redistribution of renal radiometabolites. In conclusion, monoamide derivatives of NOTA and NODAGA may be suboptimal chelators for radiocopper labeling of anti-HER2 affibody molecules and, possibly, other scaffold proteins with high renal uptake.