Leaf-level water use efficiency (WUEi) is often used to predict whole plant water use efficiency (WUEwp), however these measures rarely correlate. A better understanding of the underlying physiological relationship between WUEi and WUEwp would enable efficient phenotyping of this important plant trait to inform future crop breeding efforts. Although WUEi varies across leaf age and position, less is understood about the regulatory mechanisms. WUEi and WUEwp were determined in Australian (cv. Krichauff) and UK (cv. Gatsby) wheat cultivars. Leaf gas exchange was measured as leaves aged and evaluated in relation to foliar abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) concentration, chlorophyll content and Rubisco activity. Carbon dioxide (CO2) assimilation (A) declined more rapidly as leaves aged in the lower WUEwp genotype Gatsby. Both ACC concentration and Rubisco activity declined as leaves aged, but neither explained the variation in A. Further, stomatal conductance (gs) and stomatal sensitivity to ABA were unchanged as leaves aged, therefore WUEi was lowest in Gatsby. Maintenance of A as the leaves aged in the Australian cultivar Krichauff enabled greater biomass production even as water loss continued similarly in both genotypes, resulting in higher WUEwp. © 2020 by the authors.