Understanding the broad-scale response of lake CO2 dynamics to global change is challenging because the relative importance of different controls of surface water CO2 is not known across broad geographic extents. Using geostatistical analyses of 1080 lakes in the conterminous United States, we found that lake partial pressure of CO2 (pCO2) was controlled by different chemical and biological factors related to inputs and losses of CO2 along climate, topography, geomorphology, and land use gradients. Despite weak spatial patterns in pCO2 across the study extent, there were strong regional patterns in the pCO2 driver-response relationships, i.e., in pCO2 “regulation.” Because relationships between lake CO2 and its predictors varied spatially, global models performed poorly in explaining the variability in CO2 for U.S. lakes. The geographically varying driver-response relationships of lake pCO2 reflected major landscape gradients across the study extent and pointed to the importance of regional-scale variation in pCO2 regulation. These results indicate a higher level of organization for these physically disconnected systems than previously thought and suggest that changes in climate and land use could induce shifts in the main pathways that determine the role of lakes as sources and sinks of atmospheric CO2.