Macroalgae are important components on coral reefs that underpin food webs but are also indicators of declines in coral reef condition. The sensitivity of macroalgal communities to environmental factors, including climate and pollutants from the land, is important for the future state of coral reefs. We assessed regional variation in the photosynthetic physiology of macroalgae on the Great Barrier Reef and Moreton Bay, Queensland, Australia, using δ13C, tissue chemistry, and metabolic indicators over broad environmental gradients from six sites spanning 13° of latitude and varying distance from shore. Our data set included 568 samples from 54 genera and 19 macroalgal orders. The δ13C of tissues had a strong taxonomic basis (explaining 66% of the variation in δ13C), varying significantly among algal orders. Particularly low values of δ13C, indicating direct use of CO2(aq) in photosynthesis, were mainly associated with lineages within the Rhodophyta. We observed declines in δ13C with increasing depth (inshore only) and latitude, from offshore to inshore sites and with increasing tissue N and N : P ratio. Variation in δ13C of tissues among macroalgal lineages suggests evolution of a range of CO2 acquisition pathways. Patterns in δ13C availability are most consistent with higher availability of CO2(aq) in cooler water and inshore sites. δ13C in macroalgae could provide an important tool for monitoring changes in CO2(aq) with increasing atmospheric CO2, with changes in ocean circulation and with changes to runoff from the land to the coastal oceans.