Carbon dioxide fluxes were examined over the growing seasons of 2002 and 2003 from 14 different sites in Upper Midwest (USA) to assess spatial variability of ecosystem–atmosphere CO2 exchange. These sites were exposed to similar temperature/precipitation regimes and spanned a range of vegetation types typical of the region (northern hardwood, mixed forest, red pine, jack pine, pine barrens and shrub wetland). The hardwood and red pine sites also spanned a range of stand ages (young, intermediate, mature). While seasonal changes in net ecosystem exchange (NEE) and photosynthetic parameters were coherent across the 2 years at most sites, changes in ecosystem respiration (ER) and gross ecosystem production (GEP) were not. Canopy height and vegetation type were important variables for explaining spatial variability of CO2 fluxes across the region. Light-use efficiency (LUE) was not as strongly correlated to GEP as maximum assimilation capacity (Amax). A bottom-up multi-tower land cover aggregated scaling of CO2 flux to a 2000 km2 regional flux estimate found June to August 2003 NEE, ER and GEP to be −290 ± 89, 408 ± 48, and 698 ± 73 gC m−2, respectively. Aggregated NEE, ER and GEP were 280% larger, 32% smaller and 3% larger, respectively, than that observed from a regionally integrating 447 m tall flux tower. However, when the tall tower fluxes were decomposed using a footprint-weighted influence function and then re-aggregated to a regional estimate, the resulting NEE, ER and GEP were within 11% of the multi-tower aggregation. Excluding wetland and young stand age sites from the aggregation worsened the comparison to observed fluxes. These results provide insight on the range of spatial sampling, replication, measurement error and land cover accuracy needed for multi-tiered bottom-up scaling of CO2 fluxes in heterogeneous regions such as the Upper Midwest, USA.