This study presents observations of atmospheric boundary layer CO2mole fraction from a nine-tower regional network deployed during the North American Carbon Program's Mid-Continent Intensive (MCI) during 2007–2009. The MCI region is largely agricultural, with well-documented carbon exchange available via agricultural inventories. By combining vegetation maps and tower footprints, we show the fractional influence of corn, soy, grass, and forest biomes varies widely across the MCI. Differences in the magnitude of CO2 flux from each of these biomes lead to large spatial gradients in the monthly averaged CO2mole fraction observed in the MCI. In other words, the monthly averaged gradients are tied to regional patterns in net ecosystem exchange (NEE). The daily scale gradients are more weakly connected to regional NEE, instead being governed by local weather and large-scale weather patterns. With this network of tower-based mole fraction measurements, we detect climate-driven interannual changes in crop growth that are confirmed by satellite and inventory methods. These observations show that regional-scale CO2 mole fraction networks yield large, coherent signals governed largely by regional sources and sinks of CO2.