Abstract Olive tree pruning (OTP) is an abundant and inexpensive agricultural lignocellulosic residue that is an interesting feedstock for producing bioethanol and other bio-products in the context of lignocellulosic biorefineries. However, the presence of lignin in OTP hinders the transformation processes as it limits the access to cell wall polysaccharides. On the other hand, the aromatic/phenolic structure of the lignin polymer makes it an interesting raw material for producing chemicals, fuels and other commodities that are nowadays produced from fossil fuels. Thus, the knowledge of the OTP lignin structure is crucial to develop tailor-made pretreatments for their removal as well as for additional valorization of the lignin polymer. In this work, the OTP lignin was isolated as milled wood lignin (MWL), a lignin preparation that is considered representative of the native lignin, and characterized by two-dimensional nuclear magnetic resonance (2D-NMR) and thioacidolysis. The results demonstrated that the lignin is mainly composed of guaiacyl (G) and syringyl (S) lignin units in similar abundances (S/G ratio of ~1), with minor amounts of p-hydroxyphenyl (H) units. The most abundant lignin inter-unit linkages are β-O-4′ alkyl-aryl ethers (75% of all linkages), followed by the condensed phenylcoumarans (12%) and resinols (8%), and with lower amounts of dibenzodioxocins (2%) and spirodienones (3%). The analysis of the thioacidolysis dimers gave additional information regarding the distribution of the lignin units involved in condensed interunit linkages, including 5-5′, 4-O-5′, β-5′, β-1′ and β-β′. The high lignin content (25%), together with the relatively low S/G ratio and the abundance of condensed (carbon-carbon linked) structures, points to a low reactivity of OTP lignin during delignification pretreatments.