Grassland soils are an important component of global carbon balance because of their high levels of organic matter. Over many years large areas of grassland have been converted to crop production. Consequently, soil organic matter decreased with the use of agricultural management practices, particularly tillage. Losses of soil organic matter are related with a decline in soil structure, such as soil aggregation. The relationship between soil structure and soil organic matter is integral to the C sequestration capacity of agricultural soils. Reestablishment of perennial grasses for biomass production on agricultural lands has many benefits including increasing soil organic matter and minimizing decomposition of soil organic matter through the use of minimum tillage. The objective of this research was to estimate potential C sequestration and soil structure development with the reestablishment of warm- and cool-season grasses on previously cropped land. Experimental treatments included three grass species and fallow. Switchgrass (Panicum Virgatum L.), big bluestem (Andropogon gerardii), and intermediate wheatgrass (Thinopyrum intermedium) were established in 2000 at the USDA-ARS Research Farm near Brookings, SD. Fallow plots were mowed off twice per year for weed control. Soil cores (3.1 cm ID) were taken from 0.0 to 10.0 and 10.0 to 20.0 cm depths for bulk density. Additional soil samples were collected from the topsoil (0.0 to 20.0 cm) close to the cores with a spade to determine soil organic C distribution in aggregates during June 2005. Soil samples were air-dried after removal of visible litter and plant roots from wet samples. Air-dried soil samples were separated into five aggregate sizes classes (<0.59, 0.59 to 1.00, 1.00 to 2.00, 2.00 to 4.00, and >4.00 mm diam.) using the dry-sieving method with a flat-sieve shaker. Soil organic C, total-N, particulate organic C, and root content of each aggregate size class and total-C and total–N in root biomass were determined. The concentration of organic C in grass soils was not different from that in the fallow. The concentration of particulate organic C was highest in the soil in which intermediate wheatgrass was planted, but there was no difference among other treatments. Wet aggregate stability was significantly higher and soil bulk density significantly lower in grassland soils compared to the fallow treatment. There were large differences in aggregate size distribution among grassland soils. Grassland soils had more aggregates between 2.00 to 4.00 mm and >4.00 mm size classes compared to fallow while the fallowed soil had more aggregates <0.59 mm in size. The amount of aggregates in 0.59 to 1.00 and 1.00 to 2.00 mm size classes was not different among grass species. For soil aggregates of 2.00 to 4.00mm and >4.00 mm size classes, amounts were highest for big bluestem followed by switchgrass, intermediate wheatgrass, and fallow. Root content in each aggregate size class was significantly higher in grass soils compared to fallow. However, organic C content in each aggregate size class was not different among treatments. After 4 years of grass growth, root biomass was significantly higher in grass treatments compared to fallow, but soil organic carbon did not change significantly. Carbon content of root biomass accounted for less than 6% of soil organic C and for about 30% of particulate organic C. Our results indicate that the measurement of root biomass is very important when estimating potential C sequestration of grasslands over a relatively short time period. Estimated C sequestration by root biomass trapped in the aggregates in the top 20 cm during the first four years of reestablishment was 0.46, 0.73, and 0.55 Mg C ha^-1 yr^-1 for switchgrass, intermediate wheat grass, and big bluestem, respectively.