The structure of lignosulphonates (LS) from acidic magnesium-based sulphite pulping of Eucalyptus globulus wood has been studied. LS from thin and thick spent liquors were purified by dialysis and characterised by 1D/2D Nuclear Magnetic Resonance (NMR) and Electrospray Ionization Mass Spectrometry (ESI-MS). It was suggested that major part of LS is comprised of partially sulphonated low molecular weight oligomers formed via extensive cleavage of β-O-4 bonds in eucalypt lignin upon sulphite pulping. More than ten types of LS structures derived from different lignin structural units have been identified. A significant proportion of LS (> 20%) is comprised by monomeric compounds, mainly 4-allyl-2,6-dimethoxyphenyl-α -sulphonic and 4-propyl-2,6-dimethoxyphenyl-α,γ -disulphonic acids. LS revealed partial degradation during the thin liquor evaporation leading to cleavage of ether and carbon-carbon linkages in the side chain and to the increasing of the condensation degree.