The Midwest region of the U.S.A. is one of the world's largest areas of corn and soybeans production. Prior to the transformation to modern intensive corn/soybean production, this region was previously dominated by natural tallgrass prairie vegetation. Currently only remnants of native prairies remain throughout this region. Future predictions suggest that plant production in the Midwest may be limited by precipitation and temperature due to climate change. Cross-biome long-term studies in situ are needed to understand carbon assimilation and impact of climate change on the entire region. In this study, we investigated the differences of gross primary production (GPP) and net ecosystem production (NEP) among typical (agro-) ecosystems of corn, soybean and tallgrass prairie using data from eddy flux stations from 2006 2015. Corn had the highest average annual GPP and NEP with 1321 and 328 g C m-2 yr-1, while soybean had the lowest GPP and NEP with 640 and -39 g C m-2 yr-1, respectively. Corn and soybean NEP were linearly related (p < 0.05) to leaf area index (LAI), height and phenological stage, confirming the strong coupling between plant growth and ecosystem carbon balance. Tallgrass prairie had average values of GPP and NEP of 928 and 50 g C m-2 yr-1, which was considerably lower than corn but higher than soybean. Values for GPP and NEP were reflected in inherent water use efficiency (IWUE*) and light use efficiency (LUE) among the agroecosystems. In addition, IWUE*, LUE or GPP of crops and tallgrass prairie were also found to be linearly related (p < 0.05) to precipitation, volumetric water content (VWC) and maximum air temperature. Air temperature increased IWUE* in both, cropland and prairie vegetation. However, rainfall and soil VWC affected crops and prairie vegetation differently. While excessive rainfall and VWC reduced GPP or IWUE* in cropland, prairie vegetation GPP and LUE increased with increasing precipitation and soil VWC. Future monitoring of climate change impacts and adaptation should consider the variable effects of precipitation and temperature among the different agro-ecosystems of the Midwest.