The carbon isotope composition (δ13 C) of terrestrial plant biomarkers, such as leaf waxes and terpenoids, provides insights into past carbon cycling. The δ13 C values of modern plant biomarkers are known to be sensitive to climate and vegetation type, both of which influence fractionation during lipid biosynthesis by altering plant carbon supply and its biochemical allocation. It is not known if fractionation observed in living plants can be used to interpret fossil lipids because plant biochemical characteristics may have evolved during the Cenozoic in response to changes in global climate and atmospheric CO2. The goal of this study was to determine if fractionation during photosynthesis (Δleaf) in the Paleogene was consistent with expectations based on living plants. To study plant fractionation during the Paleogene, we collected samples from eight stratigraphic beds in the Bighorn Basin (Wyoming, USA) that ranged in age from 63 to 53 Ma. For each sample, we measured the δ13 C of angiosperm biomarkers (triterpenoids and n-alkanes) and, abundance permitting, conifer biomarkers (diterpenoids). Leaf δ13 C values estimated from different angiosperms biomarkers were consistently 2‰ lower than leaf δ13 C values for conifers calculated from diterpenoids. This difference is consistent with observations of living conifers and angiosperms and the consistency among different biomarkers suggests ancient εlipid values were similar to those in living plants. From these biomarker-based δ13Cleaf values and independent records of atmospheric δ13 C values, we calculated Δleaf. These calculated Δleaf values were then compared to Δleaf values modeled by applying the effects that precipitation and major taxonomic group in living plants have on Δleaf values. Calculated and modeled Δleaf values were offset by less than a permil. This similarity suggests that carbon fractionation in Paleogene plants changed with water availability and major taxonomic group to about the same degree it does today. Further, paleoproxy data suggest at least two of the stratigraphic beds were deposited at times when pCO2 levels were higher than today. Biomarker data from these beds are not consistent with elevated Δleaf values, possibly because plants adapted carbon uptake and assimilation characteristics to pCO2 changes over long timescales.