Water deficit induces reactive oxygen species (ROS) overproduction, which in turn inhibits plant growth and development. High concentrations of ROS disrupt the osmotic balance in plant cells and alter membrane integrity. Chromosomes carrying structural or regulatory genes must be detected to better understand plant response mechanisms to stress. The aim of our study was to identify Triticum aestivum L. chromosomes involved in early responses to short-term water-deficit stress (1, 3 and 6 h). In the present study, intervarietal substitution lines of drought-tolerant 'Saratovskaya 29' and sensitive 'Janetzkis Probat' wheat cultivars were examined. We studied the biochemical plant response system and conducted an analysis of catalase, ascorbate peroxidase and guaiacol peroxidase activities, levels of lipid peroxidation and changes in relative water content. Our results determined that the first reaction was a significant increase in guaiacol peroxidase (GPX) activity. However, the strongest impact on plant responses was found for catalase (CAT), which caused a significant decrease in lipid peroxidation (LPO) levels. Our findings indicate that chromosomes 5A, 4B, 6B and 7D are associated with early responses to short-term osmotic stress in wheat.