Abstract: Management practices can influence both the quantity of soil organic carbon (SOC) and its distribution into different fractions or pools. We investigated SOC sequestration potentials of cropping systems in near-surface (0–5 cm) samples through soil size and density fractionation coupled with acid hydrolysis and natural abundance of stable isotopes (δ13C) in a 20-year field study in the southern Great Plains in 2002. Treatments included two tillage regimens, conventional and no tillage (NT), in combination with two cropping systems: continuous grain sorghum [Sorghum bicolor (L.) Moench.] (CS) and a sorghum-wheat (Triticum aestivum L.)-soybean [Glycine max (L.) Merr.] (SWSoy) rotation, and with or without N fertilization. Tillage and cropping sequence significantly affected SOC distribution and the natural abundance of δ13C in different fractions. Samples from CS exhibited δ13C values ranging from −15‰ to −20‰, suggesting most SOC was derived from this C4 crop species. The δ13C values for soils from SWSoy varied from −20‰ to −22‰, reflecting a mixed input from C3-derived and C4-derived residue input. For whole soil and all aggregate-size fractions, SOC concentrations were significantly higher for NT than conventional tillage. However, the effects of cropping system and N fertilization on SOC interacted with tillage. Greater SOC for enhanced cropping (SWSoy) or N fertilization was observed only under NT. The fraction of <53 μm represented a greater proportion of soil than other aggregate-size fractions. Our long-term study indicated that SOC and its various fractions, including more resistant, can be increased by NT with enhanced cropping and N fertilization.