Rivers, representing the primary conduits of dissolved inorganic carbon (DIC) from the continents to the oceans, are important components to the global carbon cycle. To better understand the complex carbon cycling dynamics within two nested, mixed lithology watersheds, two sites were studied along the karst influenced upper Green River in south‐central Kentucky, USA. Weekly samples were collected from June 2013 through May 2014 and analyzed for δ13CDIC. The mixing model IsoSource was employed to better understand source partitioning differences over seasonal time spans and across the two nested basins. In both the lithologically mixed upstream basin (53% carbonate rocks, 47% siliciclastic) and carbonate rock dominated downstream basin (96% carbonate rocks in the drainage area between Greensburg and Munfordville, 78% in the total area upstream from Munfordville), DIC was primarily derived from soil respiration. The proportion of DIC from dissolved carbonate minerals derived from the downstream carbonate rock dominated basin was similar to the upstream basin, due to carbonate mineral dissolution having such a consistent effect on the overall DIC content of the river. Seasonally, soil respiration provided the most DIC from fall to winter. Early spring precipitation, combined with limited seasonal photosynthesis, shifted groundwater to be the primary source of DIC, bringing in a flush of carbonate mineral‐rich water during higher flows. This study provides insight into carbon dynamics across multiple lithologies and the important influence of seasonality using carbon isotope sourcing to determine carbonate mineral dissolution variability and aid in understanding its contribution to global carbon flux quantification. Copyright © 2015 John Wiley & Sons, Ltd.