The micronutrient selenium (Se) exhibits a narrow range between essentiality and toxicity. In soils, Se speciation influences its mobility and plant availability, with implications for addressing unsafe Se levels in plant-based nutrition. We investigated how Se speciation varies with the molecular composition of organic matter (OM) in 92 Swiss topsoils spanning different land uses (i.e., croplands, grasslands, and forests). OM composition was characterized using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), while Se speciation was determined in NaOH extracts using size exclusion chromatography coupled to UV and elemental mass spectrometry (SEC-UV-ICP-MS/MS). We found that Se speciation strongly relates to OM decomposition status and pH, and drastically differs between soil land uses. Cropland soils exhibited higher proportions of Se oxyanions and small hydrophilic organic Se, whereas forest and grassland soils contained more larger, aromatic organic Se compounds. Overall, these larger Se forms correlated with fresh and/or poorly decomposed, plant-derived OM, while oxyanions and small hydrophilic Se were linked to decomposed OM. Additionally, Se extractability by NaOH decreased with increasing soil pH, which may be due to stronger SOM stabilization, microbial processes, or higher Ca contents at higher pH. These results have important implications for Se plant availability considering land use changes and SOM degradation.