Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter‐glacial CO2

C₄ photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C₄ plants were not conducted at the low atmospheric CO₂ prevailing during their evolution. Here, 24 C₄ grasses belonging to three biochemical subtypes nicotinamide adenine dinucleotide malic enzyme (NAD‐ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP‐ME)] and six major evolutionary lineages were grown under ambient (400 μL L^?1) and inter‐glacial (280 μL L^?1) CO₂. We hypothesized that nitrogen‐related and water‐related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO₂ treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD‐ME group, while NADP‐ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO₂ and by high conductance at inter‐glacial CO₂. In conclusion, the evolutionary and biochemical diversity among C₄ grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.