Water shortage is a major environmental stress that causes the generation of reactive oxygen species (ROS). The increase in ROS production induces molecular responses, which are key factors in determining the level of plant tolerance to stresses, including drought. The aim of this study was to determine the expression levels of genes encoding MAPKs (MAPK3 and MAPK6), antioxidant enzymes (CAT, APX and GPX) and enzymes involved in proline biosynthesis (P5CS and P5CR) in Triticum aestivum L. seedlings in response to short-term drought conditions. A series of wheat intervarietal substitution lines (ISCSLs) obtained by the substitution of single chromosomes from a drought-sensitive cultivar into the genetic background of a drought-tolerant cultivar was used. This source material allowed the chromosomal localization of the genetic elements involved in the response to the analyzed stress factor (drought). The results indicated that the initial plant response to drought stress resulted notably in changes in the expression of MAPK6 and CAT and both the P5CS and P5CR genes. Our results showed that the substitution of chromosomes 3B, 5A, 7B and 7D had the greatest impact on the expression level of all tested genes, which indicates that they contain genetic elements that have a significant function in controlling tolerance to water deficits in the wheat genome.