Abstract Between 30 and 63% of the soil organic matter (SOM) is stored below 30 cm, making subsoil-SOM an important source and sink in the global carbon cycle. Nevertheless, detailed information on the composition of subsoil-SOM remains scarce. This study aims to evaluate the chemical composition of SOM in topsoil and subsoil horizons in broadleaved forests on acid loamy soils. Six sites were chosen in Northern Belgium under beech, oak and hybrid poplar, on Gleysols, Umbrisols, Cambisols and Albeluvisols on loamy Quaternary deposits. Analytical pyrolysis–gas chromatography/mass spectrometry (pyrolysis–GC/MS) was performed on the dialyzed alkaline extract, which represents between 41 and 90% of the total organic carbon for the selected sites. All extracts show a significant shift in chemical composition between the topsoil and the subsoil. While topsoil-SOM mainly differs according to input and nutrient status, subsoil-SOM shows high relative amounts of alkanes and alkenes or polysaccharides for coarse and fine textured soils respectively. Lignins, lignin-derived phenols or aromatics were not major contributors to subsoil-SOM, regardless of soil type. Furthermore, results show that very labile plant-derived molecules are present in the subsoil, i.e. long-chain aliphatics and (cellulose-derived) anhydrosugars. The organic matter signature of the subsoil samples was evaluated for typical indications of fresh material, decay, podzolisation and anaerobic processes, and indicates root input and stabilization of certain labile plant-derived compounds against microbial decay to be important in the subsoil.