AbstractMesophyll conductance (gm) is an important factor limiting photosynthesis. However, gm 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 gm under current environmental conditions, in four [CO2] treatments: glacial (180 μmol mol−1), pre-industrial (280 μmol mol−1), current ambient (450 μmol mol−1) and super-elevated (1000 μmol mol−1) in well-watered and moderate water limitation conditions, to develop an evolutionary and future climate perspective on gm control of photosynthesis and water use efficiency (WUE). In the two wheat genotypes, gm increased with rising [CO2] from glacial to ambient [CO2], but declined at super-elevated [CO2]; however, the specific mechanism of gm response to [CO2] remains unclear. Although gm and gm/gsc (mesophyll conductance/stomatal conductance) were strongly associated with the variability of A and WUE, we found that plants with higher gm may increase A without increasing gsc, which increased WUE. These results may be useful to inform plant breeding programs and cultivar selection for Australian wheat under future environmental conditions.HighlightMesophyll conductance increased with increasing [CO2] from glacial to ambient CO2 levels, then declined at super-elevated CO2 for both well-watered and water-limited treatments. These responses of mesophyll conductance with varying [CO2] have a physiological basis.