Despite the growing importance of Eucalyptus wood as raw material for pulp and paper, there is a lack of knowledge on the chemistry of their macromolecular components. The present paper addresses this issue by applying the recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) from Eucalyptus grandis, Eucalyptus globulus and the softwood species Douglas fir and white fir, which were used for comparative purposes. The structures of EMALs were investigated by quantitative P-31 NMR, DFRC/P-31 NMR (derivatization followed by reductive cleavage followed by quantitative P-31 NMR) and size exclusion chromatography (SEC). Overall, the yields of EMALs isolated from Eucalyptus were higher than those from the softwoods examined. Lignin from E globulus was found to contain higher contents of arylglycerol-beta-aryl ether structures, free phenolic hydroxyl groups and syringyl-type units than lignin from E grandis. New insights provided by the DFRC/P-31 NMR revealed that up to 62.2% of arylglycerol-beta-aryl ether structures in E globulus are uncondensed, while in E grandis the amount of such uncondensed structures was found to be lower than 48%. SEC analyses showed that lignins from E grandis and softwoods associate in greater extension than lignin from E globulus.