Abiotic stresses, such as cold, drought or high salinity, seriously affect plant growth and reduce yield in crop species including common wheat (Triticum aestivum L.). The phytohormone ABA plays important roles in plant adaptation to abiotic stress. We compared responsiveness to exogenous ABA, based on root growth inhibition by ABA, among three common wheat cultivars. Seedlings of the cultivars Cheyenne (Cnn) and Hope showed higher ABA responsiveness and higher levels of Cor (cold-responsive)-Lea (late embryogenesis abundant) gene expression than seedlings of Chinese Spring (CS). The chromosomes involved in the regulation of ABA responsiveness and Cor-Lea expression were identified using chromosome substitution lines, in which a chromosome pair of CS was substituted for the corresponding homologous pair of Cnn or Hope. In the CS-Cnn substitution lines, chromosomes 3A, 5A, 5D and 7A increased the ABA responsiveness of CS. Chromosomes 3A and 5A were also involved in the regulation of Cor-Lea gene expression and stomatal response during leaf dehydration. Substitution of CS chromosomes 3A or 5A with the respective homologous pair from Hope also enhanced ABA responsiveness and Cor-Lea expression. In addition, the factors present on chromosomes 4D and 7B of highly responsive cultivars increased Wrab17 expression but had little or no effect on ABA responsiveness. Cultivar differences in ABA responsiveness appear to be determined by genes present on these specific chromosomes in common wheat.