Separation of water-soluble lignins from lignocellulosic biomass provides a new and still poorly exploited feedstock to increase the sustainability of biorefineries. We applied derivatization followed by a reductive cleavage (DFRC) method, 2D-HSQC-NMR, and ³¹PNMR after ³¹P-labeling, to investigate molecular composition in water-soluble lignins obtained by alkaline oxidation from three biomass materials for energy (miscanthus, giant reed and an industrially pre-treated giant reed). Chromatographic identification of lignin products cleaved by DFRC showed a large predominance of guaiacyl (G) units in all biomasses and a lesser abundance of syringyl (S) and p-coumaryl (P) monomers. Our S/G ratios disagree with those reported in literature by other lignin separation methods. Carboxyl functions (ferulic and pcoumaric acids) were revealed by heterocorrelated ¹H–¹³C HSQC-NMR, and confirmed by ³¹P-NMR spectra of ³¹ P-labeled lignin molecules. An understanding of molecular composition of water-soluble lignins from biomass sources for energy is essential for lignin most efficient exploitation in either industrial or agricultural applications.