Soil organic matter is considered a key attribute for a sustainable agricultural production and is influenced by the quantity and quality of the crop residue deposited on the soil surface. Therefore, different crop rotations could change the soil organic matter pools. The objectives of this study were to evaluate the soil carbon pools obtained by chemical and physical fractionation methods and the humification index under different crop rotations in a no-till system. We test the following hypothesis: a) the distribution of C and N among the soil organic matter fractions depends on plant species rotation schemes and; b) labile fractions are more sensitive to the input of crop residues and therefore, more suitable for evaluating the impact of different crop rotations in the soil organic matter quality. We evaluated four crop sequences (corn/corn/corn; corn/wheat/corn; soybean/wheat/corn and soybean/corn/corn) in a no-till system. A five-year reforested area was used as reference. We determined the total C and N contents, the mineral-associated C and N, the light fraction of C and N, the labile carbon extracted with KMnO4 and the soil organic matter humification index. We found narrow differences between the crop rotation systems in the total C and N levels, the mineral-associated C and N fractions and the labile C extracted with KMnO4. The diversification of the agricultural system with soybean in crop rotation favored the accumulation of light fraction C and N in the soil that were more efficient to provide information about the changes in the soil organic matter quality.