Various arabino-xylo-oligosaccharides with known substitution patterns were assessed by negative ESI-Q-TOFMS and ESI-ITMS. The CID spectra of linear xylo-oligosaccharides and of nine isomeric mono- and disubstituted arabino-xylo-oligosaccharides established that structures differing in their substitution pattern can be differentiated by this approach. The negative-ion fragmentation spectra of the deprotonated quasi-molecular ions are mainly characterized by glycosidic cleavage ions from the C-series, which provide sequence informations, and by cross-ring cleavage 0,2A i ions, which provide partial linkage information. When the collision energy increased, the cross-ring cleavage 0,2A i ions underwent consecutive loss of water to produce 0,2A i − 18 fragment ions and glycosidic cleavage ions of the B-series are also produced besides the C i ions. Contrary to linear xylo-oligosaccharides, C i ions, which originate from C-3 monosubstituted xylosyl residues never produce the related cross-ring cleavage 0,2A i ions. Disubstitution at O-2 and O-3 of xylosyl residues appears to enhance the production of the 0,2A i ions compared to monosubstitution. For the differentiation of the mono- and disubstitution patterns of the penultimate xylosyl residue, the relative abundance of the glycosidic cleavage ions at m/ z 263 and 299 found on Q-TOF CID spectra plays a relevant role and appears to be more informative than MS n spectra obtained on a ion trap instrument.