Terminal drought is a common abiotic stress affecting wheat yield in Mediterranean-type environments. As terminal drought develops, top layers of the soil profile dry, exposing the upper part of the root system to soil water deficit while deeper roots can still access soil water. Since open stomata rapidly exhausts available soil water, reducing stomatal conductance to prolong availability of soil water during grain filling may improve wheat yields in water-limited environments. It was hypothesised that genotypes with more root biomass in the drying upper layer of the soil profile accumulate more abscisic acid in the leaf and initiate stomatal closure to regulate water use under terminal drought. The wheat cultivar Drysdale and the breeding line IGW-3262 were grown in pots horizontally split into two segments by a wax-coated layer that hydraulically isolated the top and bottom segments, but allowed roots to grow into the bottom segment. Terminal drought was induced from anthesis by withholding water from (i) the top segment only (DW) and (ii) the top and bottom segments (DD) while both segments in well-watered pots (WW) were maintained at 90% pot soil water capacity. Drysdale, initiated stomatal closure earlier than IGW-3262, possibly due to higher signal strength generated in its relatively larger proportion of roots in the drying top segment. The relationship between leaf ABA and stomatal conductance was strong in Drysdale but weak in IGW-3262. Analysis of ABA metabolites suggests possible differences in ABA metabolism between these two genotypes. A higher capability of deeper roots to extract available water is also important in reducing the gap between actual and potential yield.