Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [Tc-99m]Tc-(HE)(3)-G3 and [Tc-99m]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [Tc-99m]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [Tc-99m]Tc-(HE)(3)-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [Tc-99m]Tc-(HE)(3)-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.