This study quantifies small-scale carbon dioxide (CO2) efflux and estimates annual CO2 emission from a headwater stream at the Konza Prairie Long-Term Ecological Research Site and Biological Station (Konza), in a terrain of horizontal, alternating limestones and shales. We characterized the CO2 efflux and stable carbon isotopes (δ13C-CO2) at point sources of groundwater discharge from small-scale karst features, identified by temperature, and downstream of those point sources, as well as in stream reaches without identifiable point sources. CO2 effluxes ranged from 2.2 to 214 g CO2 m-2 day-1 (mean was 20.9± 41.4 g CO2 m-2 day-1). Downstream of point groundwater discharge sources, CO2 efflux decreased, over 2 meters, to 3% to 40% of the point-source flux, while δ13C-CO2 increased, ranging from -9.8 ‰ to -23.2 ‰ V-PDB (mean was -14.9 ‰ ± 4.2 ‰ V-PDB). The δ13C-CO2 increase was not strictly proportional to the CO2 flux but related to the origin of vadose-zone CO2 (C3 versus C4 vegetation). Over the study period, ~7.0 metric tons of CO2 were emitted from the 1.1-km-long stream, comparable to other headwater streams. The high spatial and temporal variability of CO2 efflux from this headwater stream informs those doing similar measurements and those working on upscaling stream data, that local variability should be assessed to make the best estimate of the impact of headwater stream CO2 efflux on the global carbon cycle.