Spatio-temporal modifications of the composition of dissolved organic matter (DOM) from a wetland in an agricultural catchment were investigated using thermally assisted hydrolysis and methylation with tetramethylammonium hydroxide coupled to gas chromatography and mass spectrometry (THM-GC–MS). The results were compared with previous data acquired on the same samples using ultraviolet spectroscopy and the stable C isotope ratio. The correlation between molecular markers and bulk-scale descriptors validated the use of THM-GC–MS to investigate the fate of DOM in soils. Molecular proxies, including lignin markers, tannin markers, carbohydrates, and fatty acids, were classified into plant-derived and microbial markers. At the beginning of the hydrologic year, associated with the recharge period, the DOM composition was similar along the depth profile and >70% of the analyzed markers were from plant-derived origins. The rise in the water table was associated with a shift from vertical to horizontal water flow due to water saturation and resulted in a clear vertical heterogeneity, i.e., a varying composition throughout the profile. In the surface horizons, the proportion of plant-derived markers remained >70%, while in the deep horizon this proportion was <30%. Last, the lowering of the water table resulted in the homogenization of the DOM composition along the depth profile.