Individual molecular components of soil organic matter (SOM) have been studied since the early 19th century. Their characterization is essential, because knowledge of the molecular structures constituting SOM allows for the detailed understanding of its origin and the processes implicated in soil C sequestration. This provides the basis for target-oriented development of management practices to optimize ecosystem services provided by soil. The aim of this review is to give an overview about the major analytical developments and the information gain that could be achieved by application of molecular methods in SOM research. Up to the 1980s, studies concentrated on the characterization of the chemical nature of SOM. Analyses were mainly based on soluble OM compounds, which were fractionated chemically into humic and fulvic acids. During the 1990s, the focus changed and scientists were more interested in biological processes controlling SOM formation and its dynamics. Introduction of physical fractionation and combination of molecular and isotopic techniques allowed assessment of composition, origin, and turnover of SOM within specific localizations in the mineral soil matrix. Analyses of the dynamics of single molecules led to a massive change of paradigms. Long residence times of SOM are no longer explained by chemical recalcitrance but by microbial products being stabilized by the interaction with soil minerals and microbial inaccessibility. In recent years, techniques yielding results with high molecular and spatial resolution were introduced, which will allow the acquisition of much more detailed information, moving a step further toward elucidating the nature and properties of SOM.