Electrospray ionization ion trap mass spectrometry has been used to distinguish three pairs of positional isomers of a new series of N-blocked hybrid peptides derived from repeats of phenylalanine(D)-β3-h-valine/β3-h-valine-phenylalanine(D) (FβV/βVF) non-natural amino acids. MSn of protonated isomeric peptides produces characteristic fragmentation involving the peptide backbone, the Boc group and the side chain. FβV-peptides can be distinguished from βVF-peptides by the loss of R-OH from [M+H−Boc+H]+, which is either of relatively low abundance or totally absent for the latter peptides. In contrast, βVF-peptides show abundant Mannich base characteristic ions by the elimination of ammonia, and imine due to a retro-Mannich cleavage. This fragmentation is absent for FβV-peptides. When β-valine is at the C-terminus, abundant b ions are produced. This is ascribed to the probable formation of a stable diketopiperazine structure, and this has been supported by the loss of H2O and CO in the CID spectra of b ions. The hybrid dipeptide acids have also been distinguished in negative ion mass spectrometry. Copyright © 2007 John Wiley & Sons, Ltd.