The present study shows the development of 99mTc-labeling of the P149-QY peptide, a modified fragment of human alpha-fetoprotein (hAFP), with the intention of potential application for diagnosis of AFP positive tumors. An efficient preparation of 99mTc-labeled P149-QY using HYNIC was developed. It has been shown that the free sulfhydryl groups in P149-QY can be protected with N-ethylmaleimide, preventing the radiolabeled peptide from loss of its biological activity. The biodistribution studies revealed favorable pharmacokinetics and a better specificity to the mammary adenocarcinoma tumor for the SH-protected radiolabeled P149-QY peptide in comparison to the radioiodinated and to the directly 99mTc-labeled peptide.