Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [Tc-99m]Tc-(HE)(3)-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)(3)), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G(3)C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)(3)-linker (designated as G3-(G(3)S)(3)C) would further improve the contrast of imaging using Tc-99m-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [Tc-99m]Tc-G3-G(3)C, [Tc-99m]Tc-G3-(G(3)S)(3)C, and [Tc-99m]Tc-G3-E3C in mice was compared with the biodistribution of [Tc-99m]Tc-(HE)(3)-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [Tc-99m]Tc-(HE)(3)-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.