Radionuclide molecular imaging of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal cancer might be used to stratify patients for HER2-targeted therapy as well as monitor treatment response and disease progression. Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins with favorable properties for molecular imaging. Herein we compared two methods for labeling the anti-HER2 DARPin (HE)(3)-G3, direct and indirect radioiodination. We hypothesized that the use of N-succinimidyl-para-iodobenzoate (SPIB) for radioiodination would facilitate the clearance of radiometabolites and improve the contrast of imaging. Both radiolabeled (HE)(3)-G3 variants preserved their binding specificity and high affinity to HER2-expressing cells. The specificity of tumor targeting in vivo was also demonstrated. A biodistribution comparison of [I-125]I-(HE)(3)-G3 and [I-125]I-PIB-(HE)(3)-G3, in mice bearing HER2 expressing SKOV3 xenografts, showed rapid clearance of [I-125]I-PIB-(HE)(3)-G3 from normal organs and tissues and low accumulation of activity in organs with NaI-symporter expression. Both radiolabeled (HE)(3)-G3 variants had equal tumor uptake. Consequently, the indirect label provided higher tumor-to-blood and tumor-to-organ ratios compared with the direct label. Comparative Single Photon Emission Computed Tomography (SPECT)/CT imaging of HER2 expression in SKOV3 xenografts, using both radiolabeled DARPins, demonstrated the superior imaging contrast of the indirect label. Indirect radioiodination of (HE)(3)-G3 using SPIB could be further applied for SPECT and PET imaging with iodine-123 and iodine-124.