Low molecular weight (LMW) biomarkers can be used to trace the source and stage of soil organic matter. However, methods that selectively isolate these groups of compounds are underdeveloped. In this study, we isolate biomarkers by a successive series of extraction and chemical degradation procedures involving solvent extraction (TSE), base (BHY) and acid hydrolysis (AHY), and CuO oxidation (CUO). GC-MS was used to analyze these fractions and the extraction methods were verified by solid-state 13C NMR spectroscopy. The GC-MS response was high for the BHY products (96%), intermediate for the TSE (30%) and CUO (19%), but very low for the AHY fraction (5%) indicating that the fractions contain polar or high molecular weight compounds. Aliphatic lipids (62%), phenols and benzyls (17%) were the predominant classes, accompanied by minor abundances of mono- and disaccharides, LMW acids, terpenoids, steroids, amino acids, and amino sugars. The TSE and BHY fractions contained mainly aliphatic lipids derived from plant waxes, cutin, and suberin. Lignin-derived phenols are the major products in the CUO fraction, and amino compounds and carbohydrates of various sources were identified in the AHY products. The sequential degradation method is useful for the isolation and identification of apolar, LMW biomarkers in soil.