Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARPin G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)3) of histidine-containing tags would influence the biodistribution of [99mTc]Tc(CO)3-labeled DARPin G3. To test the hypothesis, G3 variants containing tags at N-terminus (H6-G3 and (HE)3-G3) or at C-terminus (G3-H6 and G3-(HE)3) were labeled with [99mTc]Tc(CO)3. Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [99mTc]Tc(CO)3-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [99mTc]Tc(CO)3-(HE)3-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [99mTc]Tc(CO)3-(HE)3-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.