Technical Abstract: We carried out mid-infrared (MidIR) spectral interpretation of fractionated fresh and incubated agricultural soils to determine changes in soil organic matter (SOM) chemistry during long-term incubation. Soils cores from four long-term sites under continuous corn in the US Corn Belt were obtained from 0-20, 25-50, and 50-80 cm depth. The samples were then subject to 440 d and 800 d of laboratory incubation. At each sampling time, the soils were processed to obtain the light fraction (LF), particulate organic matter (POM), silt-sized (silt), and clay-sized (clay) fractions. Time zero samples were also acidified to obtain decalcified soils. The dried and ground samples were scanned as neat samples in diffuse reflectance from 400 to 4000 cm-1. The objectives were to determine the differences in MidIR spectral properties between sites, depths, incubated samples, and fractions. Our results show that the LF and clay fractions have distinct spectral features regardless of site, suggesting that there is a common chemistry for each of these C pools. The LF is characterized by OH/NH band at 3400 cm-1, as well as carboxylic, protein, amides, aliphatic and aromatic C bond absorbance between 1350-1750 cm-1. The clay fraction is distinguished by aromatic ring CH absorption near 1230 cm-1, and absorption at 780-620 cm-1. Lamberton and Hoytville soils, which are high in organic C, are also characterized by absorbance at 1230 cm-1, suggesting that this band marks the presence of high SOM. In turn, the POM, silt and whole soil have different spectral properties from the LF or clay. The POM, like the LF, absorbs at the broad peak at 1360 cm-1 that encompasses CH2, CH3, COO, COOH in aliphatics and phenolics, as well as amines. Soils from different sampling depths also have specific spectral properties. The CH bending region between 1370-1450 cm-1 is characteristic of shallow depths. Because of their low OM content, the deeper samples are characterized by mineral features such as quartz, clays and calcite/dolomite. Spectroscopic data indicates that the clay fraction and the LF suffered measurable chemical transformations during the 800 d incubation, but the POM and Silt fraction did not. As the LF decomposes, it loses OH/N-H bonds (3400 cm-1), and aromatic and aliphatic CH bonds (1223 cm-1, 2920-2860 cm-1). The clay fraction suffered changes that were opposite to those of the LF, indicating that LF decomposition and clay decomposition follow different chemistries.