The lignin from Cynara cardunculus stalks was isolated by the classical Björkman method and characterized by pyrolysis coupled with gas chromatography and mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and derivatization followed by reductive cleavage (DFRC). The milled Cynara lignin (MCyL) was constituted mainly by guaiacyl (G) and syringyl-units(S) (S/G molar ratio of 0.7), with the complete absence of p-hydroxyphenyl (H) units. The 2D-NMR analysis indicated a predominance of alkyl-aryl ether linkages (70 % of all inter unit linkages are β–O–4′) and significant amounts of condensed structures such as phenylcoumarans (β-5′, 14 %), resinols (β-β′, 7 %), spirodienones (β-1′, 5 %), and dibenzodioxocins (5-5′, 4 %). Furthermore, the analyses indicated that the lignin is partially acylated at the γ-OH (12 % acylation) by acetate groups and that acetylation occurs preferentially on syringyl-units. As in other plants, acetylation occurs at the monomer stage, and sinapyl acetate behaves as a real lignin monomer participating in lignification in cardoon stalks. The detailed structural characterization of cardoon lignin reported here will foster the industrial use of this biomass for the production of biofuels and other bio-based chemicals under the lignocellulosic biorefinery.