Mesophyll conductance (g m) is an important factor limiting photosynthesis. However, g m response to long-term growth in variable [CO2] is not well understood, particularly in crop plants. Here, we grew two cultivars of wheat (Halberd and Cranbrook), known to differ in g m under current environmental conditions, in four [CO2] treatments: glacial (206 μmol mol−1), pre-industrial (344 μmol mol−1), current ambient (489 μmol mol−1), and super-elevated (1085 μmol mol−1), and two water treatments (well-watered and moderate water limitation), to develop an evolutionary and future climate perspective on g m control of photosynthesis and water-use efficiency (WUE). In the two wheat genotypes, g m increased with rising [CO2] from glacial to ambient [CO2], but declined at super-elevated [CO2]. The responses of g m to different growth [CO2] also depend on water stress; however, the specific mechanism of g m response to [CO2] remains unclear. Although g m and g m/ g sc (mesophyll conductance/stomatal conductance) were strongly associated with the variability of photosynthetic rates (A) and WUE, we found that plants with higher g m may increase A without increasing g sc, which increased WUE. These results may be useful to inform plant breeding programmes and cultivar selection for Australian wheat under future environmental conditions. [ABSTRACT FROM AUTHOR]