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This study tested the possible root biomass improvements in crop rotations after the conversion of grasslands, and crop samples from maize, winter wheat, and winter barley were collected during 2011–2013 from a long-term experimental site in Lusignan, France (http://www.soere-acbb). Root biomass C quantification was performed using δ13C isotopic signatures to determine the presence of both C3 and C4 plants. We also calculated the recovery rate of maize root biomass C. The results showed that after crop rotations, 0–60 cm root biomass C values were 44.1, 34.2, and 18.7 g C m−2 for maize, winter wheat, and winter barley respectively. The Root biomass C of crops after conversion to grassland was approximately 2–3 times those observed after crop rotations. However, incorporating ley grassland duration into crop rotations showed limited improvements in shoot biomass C and grain yield of the crops, regardless of the decreased rate of N fertilizer for maize. Moreover, root biomass C had a significant relationship with N supply from residues (P <.05). Nevertheless, shoot biomass C of only maize showed significance in its relationships with N supply and root biomass C. In addition, in each 30 cm soil layer (0–30 cm, 30–60 cm, and 60–90 cm), the recovery rate of maize roots decreased to approximately 15% when root biomass C increased to 10 g C m−2. However, further increases in root biomass C had little impact on the recovery rate. In conclusion, compared with continuous cropland, incorporating ley grassland duration into crop rotations increases root biomass C of crops, but this change may not be a significant increase of the shoot biomass C or grain yield. This finding simply indicates the improved C input from crops and the potential to increase soil organic C, as well as providing a model for the sustainability of crop rotations. Unlabelled Image • Incorporating ley grassland into crop rotation increased residual N supply for crops. • Incorporating ley grassland into crop rotation increased root biomass C of crops. • Incorporating ley grassland in crop rotation may not improve shoot biomass C of crops. • Variations of shoot biomass C may not reflect the variations of root biomass C. • If root biomass C are large enough, their recovery rates are close to a constant. [ABSTRACT FROM AUTHOR]