Epidermal growth factor receptors (EGFRs) are overexpressed in a variety of epithelial cancers such as epithelial ovarian cancer. In the present study the use of 99mTc-EDDA/tricine-HYNIC-(Ser)3-GE11, as a new EGFR targeting ligand for tumor imaging was investigated. HYNIC-(Ser)3-GE11 conjugated peptide was labeled with 99mTc in the presence of EDDA/tricine as coligands. The resulting radiolabeled peptide showed high radiochemical purity and high stability in serum and buffer solution. The affinity of the radiolabeled peptide for binding to EGFR was investigated on three cell lines with different levels of EGFR expression (SKOV3, A549, and MCF-7) of which SKOV3 cells accumulated higher amounts of radioactivity and was used for further in vitro and in vivo investigations. Biodistribution study in normal female mice revealed that the radioconjugated peptide had high in vivo stability and high renal excretion. Similar biodistribution pattern was observed in mouse xenograft tumor model and % ID/g values of tumor uptake of the radiolabeled peptide at 1 and 4 h post injection were 3.64 ± 0.66 and 1.7 ± 0.42%, respectively. Pre-saturation of EGFRs by excess molar amounts of unlabeled peptide significantly decreased tumor uptake value at 4 h post injection to 1.07 ± 0.17% ID/g indicating tumor specificity of the radiolabeled peptide. Also, the gamma scintigraphy showed good tumor visualization at 1 h post injection. From results of this investigation it appears that 99mTc-EDDA/tricine-HYNIC-(Ser)3-GE11 might be a promising radiotracer for imaging of tumors with high EGFR overexpression.