The stable carbon (C) isotope variability of dissolved inorganic and organic C (DIC and DOC), particulate organic carbon (POC), glucose and polar-lipid derived fatty acids (PLFAs) was studied in a survey of 22 North American oligotrophic to eutrophic lakes. The δ¹³C of different PLFAs were used as proxy for phytoplankton producers and bacterial consumers. Lake pCO₂ was primarily determined by autochthonous production (phytoplankton biomass), especially in eutrophic lakes, and governed the δ¹³C of DIC. All organic-carbon pools showed overall higher isotopic variability in eutrophic lakes (n = 11) compared to oligo-mesotrophic lakes (n = 11) because of the high variability in δ¹³C at the base of the food web (both autochthonous and allochthonous carbon). Phytoplankton δ¹³C was negatively related to lake pCO₂ over all lakes and positively related to phytoplankton biomass in eutrophic lakes, which was also reflected in a large range in photosynthetic isotope fractionation (ϵ_CO2-phyto, 8–25‰). The carbon isotope ratio of allochthonous carbon in oligo-mesotrophic lakes was rather constant, while it varied in eutrophic lakes because of maize cultivation in the watershed.