<p>Drought stress limits global wheat production, necessitating efficient evaluation of genetic resources for tolerance. Soil-based pot and polyethylene glycol (PEG)-induced hydroponic systems are commonly used to evaluate drought stress tolerance; however, the consistency of these systems in detecting genetic variation remains uncertain. Here, we evaluated genetic variation in physiological, morphological, and biochemical responses to drought in 24 Asian wheat lines using both systems. Soil plant analysis development (SPAD) values, drought tolerance index (DTI), and shoot biomass were measured before and after stress treatments. Both soil-based drought and PEG-induced osmotic stress significantly reduced SPAD and DTI values across genotypes, indicating chlorophyll loss and growth inhibition. Based on these traits, four tolerant and four susceptible lines were selected. In these lines, abscisic acid (ABA), hydrogen peroxide (H₂O₂), and γ-aminobutyric acid (GABA) levels were quantified. In both systems, ABA, H₂O₂, and GABA levels increased significantly following stress, reflecting genotype-dependent oxidative and metabolic responses. Although ABA accumulation tended to be higher under soil-based drought conditions, overall response patterns were similar between systems. The accumulation of these biochemical ccompounds was negatively correlated with SPAD and DTI values. These results highlight substantial genetic variation in drought-responsive traits among Asian wheat germplasm and indicate that hydroponic screening can effectively capture key physiological and biochemical responses associated with soil-based drought. This approach provides a practical and efficient tool for large-scale preliminary screening of wheat genetic resources and supports the identification of drought-tolerant germplasm for breeding.</p>