Objective: The aim of this study was to develop an improved method for labelling Z(HER2:342) with Technetium-99m (Tc-99m) using Gly-(D) Ala-Gly-Gly as a chelator and to evaluate the feasibility of its use for visualization of HER2 expression in vivo. Methods: The Affibody (R) molecule Z(HER2:342) was synthesized by Fmoc/tBu solid phase synthesis. The chelator, Gly-(D) Ala-Gly-Gly, was introduced by manual synthesis as the N-terminal extensions of Z(HER2:342). Z(HER2:342) was labelled with Tc-99m. The labelling efficiency, radiochemical purity and in vitro stability of the labelled molecular probe were analysed by reversed-phase high performance liquid chromatography. Biodistribution and molecular imaging using Tc-99m-peptide-Z(HER2:342) were performed. Results: The molecular probe was successfully synthesized and labelled with Tc-99m with the labelling efficiency of 98.10 +/- 1.73% (n = 5). The radiolabelled molecular probe remained highly stable in vitro. The molecular imaging showed high uptake in HER2-expressing SKOV3 xenografts, whereas the MDA-MB-231 xenografts with low HER2 expression were not clearly imaged at any time after the injection of Tc-99m-peptide-Z(HER2:342). The predominant clearance pathway for Tc-99m-peptide-Z(HER2:342) was through the kidneys. Conculsion: Tc-99m-peptide-Z(HER2:342) using Gly-(D) AlaGly- Gly as a chelator is a promising tracer agent with favourable biodistribution and imaging properties that may be developed as a radiopharmaceutical for the detection of HER2-positive malignant tumours. Advances in knowledge: The Tc-99m-peptide-Z(HER2:342) molecular probe is a promising tracer agent, and the results in this study provide a foundation for future development of protocols for earlier visual detection of cancer in the clinical setting.