CO2 diffusion from substomatal intercellular cavities to sites of carboxylation in chloroplasts (mesophyll conductance; gm) limits photosynthetic rate and influences leaf intrinsic water-use efficiency (A/gsw). We investigated genotypic variability of gm and effects of gm on A/gsw among eleven wheat (Triticum aestivum L.) genotypes under light-saturated conditions and at either 2 or 21% O2. Significant variation in gm and A/gsw was found between genotypes at both O2 concentrations, but there was no significant effect of O2 concentration on gm. Further, gm was correlated with photosynthetic rate among the 11 genotypes, but was unrelated to stomatal conductance. The effect of leaf age differed between genotypes, with gm being lower in older leaves for one genotype but not another. This study demonstrates a high level of variation in gm between wheat genotypes; 0.5 to 1.0 µmol m-2 s-1 bar-1. Further, leaf age effects indicate that great care must be taken to choose suitable leaves in studies of genotypic variation in gm and water-use efficiency.