In this study, the seedlings of two wheat cul-tivars were used: drought-resistant Chinese Spring (CS) and drought-susceptible (SQ1). Seedlings were subjected to osmotic stress in order to assess the differences in response to drought stress between resistant and susceptible genotype. The aim of the experiment was to evaluate the changes in physiological and biochemical characteristics and to establish the optimum osmotic stress level in which differences in drought resistance between the genotypes could be revealed. Plants were subjected to osmotic stress by supplementing the root medium with three concentra-tions of PEG 6000. Seedlings were grown for 21 days in control conditions and then the plants were subjected to osmotic stress for 7 days by supplementing the root med-ium with three concentrations of PEG 6000 (D1, D2, D3) applied in two steps: during the first 3 days of treatment -0.50, -0.75 and -1.00 and next -0.75, -1.25 and -1.5 MPa, respectively. Measurements of gas exchange parameters, chlorophyll content, height of seedlings, length of root, leaf and root water content, leaf osmotic potential, lipid peroxidation, and contents of soluble carbohydrates and proline were taken. The results highlighted statistically significant differences in most traits for treatment D2 and emphasized that these conditions were optimum for expressing differences in the responses to osmotic stress between SQ1 and CS wheat genotypes. The level of osmotic stress defined in this study as most suitable for differentiating drought resistance of wheat genotypes will be used in further research for genetic characterization of this trait in wheat through QTL analysis of mapping pop-ulation of doubled haploid lines derived from CS and SQ1.