Dissecting the evolutionary path to C4 photosynthesis is a critical step in engineering improvements in crop photosynthesis and yields. Three C4-acid decarboxylation enzymes (NADP-ME, NAD-ME and PEPCK) are responsible for delivering CO2 for fixation by Rubisco during C4 photosynthesis. Decarboxylase recruitment has evolved independently multiple times throughout C4 lineages. Here, a systematic yet rapid identification of key differences across species has allowed us to investigate the regulation and evolution of the three C4-acid decarboxylases in one C3 and eleven C4 grasses (Poaceae).