Fractions of soil organic matter (SOM) were obtained from three soils using alternative physical fractionation procedures, and evaluated against the requirements of model pools. We compared two-stage density fractionation (isolating free and intra-aggregate fractions, before and after dispersion, respectively) with particle-size separation of dispersed soil. For full comparison, the organomineral fraction residual from density fractionation was also size separated. In standardizing the density-based method, we found recovery of intra-aggregate organic matter highly sensitive to separation density as compared with the free. Recovery of the intra-aggregate was also influenced by dispersion energy. The greatest amount was obtained using a combination of the highest density (1.80 g cm−3) and dispersion energy (1500 J g−1). Analysis by 13C nuclear magnetic resonance (NMR) showed O-alkyl/alkyl-C ratios 1.38 to 2.30 times greater in intra-aggregate organic matter than in the free. Diffuse reflectance Fourier transform infrared spectroscopy (DRIFT) also indicated a greater proportion of aliphatic hydrocarbon, carboxylic anions, and aromatic C in intra-aggregate organic matter. The findings suggest this fraction comprises more decomposed and transformed organic matter relative to the free. Higher signal/noise ratios in NMR spectra of particle-size fractions (compared with their organomineral equivalents) were attributed to C in particulate SOM, not removed by prior density separation. Whilst particle-size fractions confuse particulate SOM with that attached to mineral surfaces, fractions isolated by two-stage density separation are small in number and display distinct chemical properties. We suggest they provide a sound basis for a model of SOM turnover based on measurable pools.