Severe iodination of a disulfide peptide molecule (peptide D) was detected from the on-resin disulfide cyclization process. LC/MS/MS results indicated that iodination occurred on the Thi (thienylalanine) residue. Systematic investigation revealed that the iodination reaction occurred predominantly in the I 2 /DMF-mediated on-resin disulfide formation reaction. The iodination side reaction was strongly solvent-dependent and favored in DMF and NMP. A tentative mechanism similar to that of the Vilsmeier−Haack reaction was proposed. A variety of amino acid/peptide substrates were tested as iodination substrates. The results indicated the susceptibility of amino acids with electron-rich aromatic groups to iodination. Additionally, various solutions were proposed for preventing peptide iodination based on the findings in this study. This study demonstrates the applicability of the onresin disulfide production of peptides bearing electron-rich aromatic residues.