The characterization of organic matter in agroecosystems is important because of its involvement in many important soil ecosystem processes. Humic acid, fulvic acid, and water-extractable organic matter from a 9-year agroecosystem study investigating the effects of tillage, cropping system, and N source was characterized using multidimensional fluorescence spectroscopy with parallel factor analysis (PARAFAC). The fluorescence spectra suite containing all three types of organic matter fractions was modeled by five PARAFAC components. The distribution of component concentrations was unique to each type of organic matter fraction, indicating that these operational extracted fractions reflect differing chemical pools of soil organic matter. The three treatment factors (tillage, cropping, and N source) investigated in this study did not affect the component distribution of the humic and fulvic acid fractions that are regarded as the refractory pools of soil organic matter. In contrast, the component distribution of the more labile water-extractable organic matter fraction was significantly affected by the N source treatment. The results of this study support the use of multidimensional fluorescence spectroscopy with PARAFAC as a method to investigate how management practices of agroecosystems affect the dynamics and chemical nature of soil organic matter pools.