Abstract: The discrimination of four pairs of cyclic peptide diastereomers were successfully achieved by ESI-MS. The ratio of ions’ abundance ratios between A and B isomers in MS/MS spectra was defined as a parameter(rA, B) for discriminating diastereomers. All the four rA, B values deviates far from 1, which demonstrates excellent discrimination of the four pairs of diastereomers. The diverse spectral behaviors of CP1-CP6 was found attributing to ‘mobile proton’ transfer diversities and steric hindrance at fragmentation sub-surrounding due to chiral differences. MSn spectra illustrate that both CP7 and CP8 fragment through bx-yz and a1-yx ring-opening mechanisms and their chiral differences result in the predilection of the two ring-opening modes.