ABSTRACT Nitrogen (N) fertilization changes both the degradation rate and chemical composition of plant litter and soil organic matter (SOM). We sought to document how N application rates and incorporation of crop straw residues in wheat-rice rotation systems are related to the chemical composition of SOM. Using Fourier transform infrared spectroscopy and ¹³C multiple cross-polarization/magic angle spinning nuclear magnetic resonance spectroscopy techniques, we investigated the chemical composition of SOM with incorporation of crop straw residues in a 6-year wheat-rice double-cropping system. Nitrogen fertilizer was applied at the rates of 0, 290, 380, and 470 kg N ha⁻¹ per year along with the incorporation of crop residues into the soil. We found that greater additions of N increased the concentrations of both C and N in the soil. The Fourier transform infrared spectra of SOM indicated that the abundance of aromatic functional groups (~1,628 and ~1,515 cm⁻¹) and the amide groups (~1,660 and ~1,550 cm⁻¹) increased in the N-amended treatments compared with the unamended treatment. ¹³C multiple cross-polarization/magic angle spinning nuclear magnetic resonance spectroscopy results revealed that the abundance of recalcitrant compounds, including alkyl and aromatic C, increased, whereas labile SOM components, dominated by O-alkyl and anomeric C, decreased with the elevated N application rates. These observations were also reflected in calculated indices of A/O-A aromaticity and hydrophobicity. Our results support the hypothesis that recalcitrant compounds of SOM appeared to be enhanced with the increase of the N application rates to promote crop straw decomposition in the rice-wheat rotation system.