Quantification of regional greenhouse gas (GHG) fluxes is essential for establishing mitigation strategies and evaluating their effectiveness. Here, we used multiple top-down approaches and multiple trace gas observations at a tall tower to estimate regional-scale GHG fluxes and evaluate the GHG fluxes derived from bottom-up approaches. We first applied the eddy covariance, equilibrium, inverse modeling (CarbonTracker), and flux aggregation methods using 3 years of carbon dioxide (CO2) measurements on a 244 m tall tower in the upper Midwest, USA. We then applied the equilibrium method for estimating CH4 and N2O fluxes with 1-month high-frequency CH4 and N2O gradient measurements on the tall tower and 1-year concentration measurements on a nearby tall tower, and evaluated the uncertainties of this application. The results indicate that (1) the flux aggregation, eddy covariance, the equilibrium method, and the CarbonTracker product all gave similar seasonal patterns of the regional CO2 flux (105−106 km2, but that the equilibrium method underestimated the July CO2 flux by 52-69%. (2) The annual budget varied among these methods from-54 to-131 g C-CO2 mg-2 yrg-1, indicating a large uncertainty in the annual CO2 flux estimation. (3) The regional CH4 and N2O emissions according to a top-down method were at least 6 and 2 times higher than the emissions from a bottom-up inventory (Emission Database for Global Atmospheric Research), respectively. (4) The global warming potentials of the CH4 and N2O emissions were equal in magnitude to the cooling benefit of the regional CO2 uptake. The regional GHG budget, including both biological and anthropogenic origins, is estimated at 7 ± 160 g CO2 equivalent mg-2 yrg-1.