Paired measurements of bulk carbonate (δ13Ccarb), organic matter (δ13Corg), and their difference (Δ13C) can be used to estimate changes in isotopic fractionation through time as a function of O2/CO2 in the atmosphere. However, because local scale processes can also affect Δ13C, it is essential to compare sections from widely separated water masses. Here we present new δ13Corg data from Ordovician carbonate rocks from the Great Basin, Oklahoma, and Appalachian Basin and compare with published δ13Ccarb records from these sections and paired δ13C values from other carbonate successions around North America. These new data complement previous studies that focused on Upper Ordovician δ13Ccarb excursions and now provide a composite Ordovician δ13Corg record. New Lower Ordovician (Tremadocian Stage) δ13Corg data range from ca. −26 to −28‰, decreasing throughout the Lower–Middle Ordovician (Floian–Dapingian Stages) to ca. −29 to −31‰. δ13Corg values remain at their lowest throughout the Sandbian and are similar to other published Upper Ordovician (Sandbian–Katian) δ13Corg data from North America. Δ13C values from well-preserved intervals generally vary between +26 to +28‰ throughout the Lower to Middle Ordovician (Tremadocian to early Darriwilian), but increase to +31‰ during the mid–late Darriwilian and mid Sandbian, similar to published data from younger Late Ordovician positive δ13C excursions known as the Guttenberg (GICE) and Hirnantian (HICE) events. The overall Δ13C trend shows a ~3‰ increase throughout the Early–Middle Ordovician and coincides with a previously interpreted period of ocean cooling and some of the earliest pulses of global biodiversity of marine invertebrates and planktonic organisms. Modeling studies predict that pCO2 decreased during this time, suggesting that the effect of pCO2 on Δ13C may have been overwhelmed by other controls, such as an in increase in pO2 or a higher O2/CO2 ratio during this biodiversification event. •New Early–Middle Ordovician paired carbon isotopic analyses are reported here.•New organic isotope data combined with published data make an Ordovician composite.•A 3‰ increase in Δ13C coincides with the onset of the GOBE and possible ocean cooling.•Cooling or pCO2 changes cannot explain the entire Δ13C increase.•Increased pO2 or atmospheric O2/CO2 ratios may explain the long-term Δ13C increase.