Membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis, and holds great promise as an early biomarker for malignant tumors. Therefore, the ability to evaluate MT1-MMP expression could be valuable for molecular biological and clinical studies. For this purpose, we aimed to develop short peptide-based nuclear probes because of their facile radiosynthesis, chemically uniform structures, and high specific activity, as compared to antibody-based probes, which could allow them to be more effective for in vivo MT1-MMP imaging. To the best of our knowledge, there have been no reports of radiolabeled peptide probes for the detection of MT1-MMP in cancer tissues. In this study, we designed and prepared four probes which consist of a MT1-MMP-specific binding peptide sequence (consisting of L or D amino acid isomers) and an additional cysteine (at the N or C-terminus) for conjugation with N-(m-[(123/125)I]iodophenyl) maleimide. We investigated probe affinity, probe stability in mice plasma, and probe biodistribution in tumor-bearing mice. Finally, in vivo micro single photon emission computed tomography (SPECT) imaging and ex vivo autoradiography were performed. Consequently, [(123)I]I-DC, a D-form peptide probe radioiodinated at the C-terminus, demonstrated greater than 1000-fold higher specific activity than previously reported antibody probes, and revealed comparably moderate binding affinity. [(125)I]I-DC showed higher stability as expected, and [(123)I]I-DC successfully identified MT1-MMP expressing tumor tissue by SPECT imaging. Furthermore, ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles corresponded to MT1-MMP-positive areas. These findings suggest that [(123)I]I-DC is a promising peptide probe for the in vivo detection of MT1-MMP in cancers.