Differentiation of biochar and native soil organic matter (SOM) is required to assess the effect of biochar amendment on in-situ changes of SOM. Therefore, we used C₄ biochar produced at high temperature (1200 °C) by gasification (BC_GS) and measured the ¹³C abundance of density and particle size fractions. We quantified the BC_GS effects on distinct native C₃-SOM pools of a grassland topsoil one year after BC_GS amendment. The chemical composition was analyzed with solid-state ¹³C CPMAS NMR, whereas information on the nanostructure of BC_GS were obtained by Raman microspectroscopy measurements. Our aim was to assess BC_GS induced chemical changes of SOM and physical fractions and to validate the accuracy of BC_GS detection by ¹³C NMR spectroscopy. Quantification by isotopic measurements and ¹³C NMR spectroscopy for aromatic C yielded similar estimates of BC in soils. Of the total BC_GS, 52% were recovered as free particulate organic matter (POM) and 33% were located in aggregated soil structures isolated as occluded POM particles. Around 4% of the total BC_GS was detected in the clay fraction. After one year of field exposure, the surface of the BC_GS particles decreased in unordered graphitic-like structures. The higher ordered BC residue is supposed to be more recalcitrant. The native SOC stock increase (p = 0.06, n = 4) in the clay fraction indicated increased sequestration of organic matter as mineral-bound SOM due to BC_GS amendment. With respect to soil functionality, the BC_GS amendment induced a tremendous shift from a soil system dominated by organo-mineral associations to POM−dominated OC storage, resulting in increased soil air capacity.