Rationale For disulfide‐containing peptides, mass spectrometric analyses are rarely comparably studied between their dithiol and disulfide forms. Persulfide ions afforded from peptides with a disulfide ring are from either an unusual N–Cα bond cleavage or a canonical peptide bond cleavage; their isomeric structures are, however, not identified just from peaks of mass spectra. Methods Isomeric structures of C3P4X5|C6M, C3MAP4X5|C6MB and P4X5C6|C3M were identified from a series of the X5 substituted dicysteine octapeptides using electrospray ionization tandem mass spectrometry for both their dithiol and disulfide forms. Formation mechanisms of different persulfide ions were investigated systematically by theoretical methods. Moreover, electrostatic potential‐mapped molecular van der Waals surfaces were used to determine the stabilities of the intermediates, which gave a further evaluation of favored bond cleavage. Results Mass spectral analyses indicated that the fragmented ions changed largely when an intramolecular disulfide bond was formed. New types of disulfide‐containing fragmented ions C3P4X5|C6M or C3MAP4X5|C6MB were thus proposed. Energy analysis showed that the N–Cα cleavage was not competitive energetically with that of the amide bond for Y5 and its phosphorylated analogue. However, the N–Cα cleavage products dominated for the S5‐ and T5‐containing peptides. Stabilities of the intermediates were found to be related with the electrostatic potential‐mapped molecular van der Waals surfaces. Conclusions Persulfide ions containing more residues than previously found were proposed not only from b7 ions but also from y6 ions. In addition, a new kind of phosphorylated analogue, C3P4pY5|C6M, is reported in this work. Our study provides convincing results for separating isomeric structures in the cases of N–Cα cleavages, which greatly assists in the structural identification of disulfide‐containing peptides.