AbstractGenetic manipulation of whole‐plant transpiration rate (TR) response to increasing atmospheric vapor pressure deficit (VPD) is a promising approach for crop adaptation to various drought regimes under current and future climates. Genotypes with a non‐linear TR response to VPD are expected to achieve yield gains under terminal drought, thanks to a water conservation strategy, while those with a linear response exhibit a consumptive strategy that is more adequate for well‐watered or transient‐drought environments. In wheat, previous efforts indicated that TR has a genetic basis under naturally fluctuating conditions, but because TR is responsive to variation in temperature, photosynthetically active radiation, and evaporative demand, the genetic basis of its response VPD per se has never been isolated. To address this, we developed a controlled‐environment gravimetric phenotyping approach where we imposed VPD regimes independent from other confounding environmental variables. We screened three nested association mapping populations totaling 150 lines, three times over a 3‐year period. The resulting dataset, based on phenotyping nearly 1400 plants, enabled constructing 63‐point response curves for each genotype, which were subjected to a genome‐wide association study. The analysis revealed a hotspot for TR response to VPD on chromosome 5A, with SNPs explaining up to 17% of the phenotypic variance. The key SNPs were found in haploblocks that are enriched in membrane‐associated genes, consistent with the hypothesized physiological determinants of the trait. These results indicate a promising potential for identifying new alleles and designing next‐gen wheat cultivars that are better adapted to current and future drought regimes.