Increasing demand for agricultural commodities such as grain for feed and feedstocks for biofuels are driving rapid land use change. We studied the effect of agricultural land-use change on two historical land use types for three different cropping systems using eddy covariance and ecosystem carbon balance approaches. We quantified the consequences of establishing corn, switchgrass, and mixed prairie species on cropland under a recent corn–soybean rotation and on set-aside land planted for 20 years to smooth bromegrass (Bromus inermis Leyss) enrolled in the USDA Conservation Reserve Program (CRP). We converted three CRP and three cropland fields to no-till soybeans in 2009 (conversion year) and then established fields of corn, switchgrass, and mixed prairie species in fields formerly cropland or CRP. In addition, one CRP perennial grassland site was kept undisturbed as a reference. We measured the harvestable biomass during the conversion and over the two following years. To account for C removed in harvestable biomass we calculated adjusted Net Ecosystem Production (NEPadj; g C m−2 yr−1) as NEP measured by eddy covariance plus harvested biomass. During the conversion year, fields converted from historical CRP grassland exhibited net C emissions between 261 and 340 g C m−2 yr−1. The sites established on recent cropland emitted 37–42 g C m−2 yr−1. The undisturbed reference site sequestered (rather than emitted) 35 g C m−2 yr−1. Cumulative NEPadj over the entire 3-year period at the sites converted from CRP grasslands was 170, 740, and 885 g C m−2 for switchgrass, corn, and mixed prairie systems, respectively. At the former cropland sites, cumulative NEPadjwas 214, 364, and 446 g C m−2 for mixed prairie, corn, and switchgrass systems, respectively. Over this period the reference site had an NEP of −260 ± 30 g C m−2. Land use change to biofuel crops thus induced large C emissions, even when renewable energy production was included as a fossil fuel carbon offset. The most productive perennial systems had lowest overall C losses. Accounting for fossil fuel offset credits generated from harvested biomass completely attenuated CO2 emissions in the CRP site converted to switchgrass and partially attenuated emissions in all other converted sites. These results can be readily incorporated into management recommendations for future establishment of biofuels feedstock and row agriculture systems with different management intensities.