Occurrences of osmotic stress have profound impacts on global wheat production. Drought may be a global issue, in any wheat-producing region that can cause severe osmotic stress. In this study, a comparative study of drought tolerance screening techniques in vitro and in vivo was conducted using fifteen winter wheat cultivars. Under in-vitro screening, for simulating drought conditions, -0.45 MPa and -0.9 MPa osmotic potential were used. Polyethylene glycol 6000 was used to induce osmotic stress and seedling traits such as germination (%), shoot length, total root length, total root number, fresh and dry weights of shoots and roots, also proline content were studied. In the case of in vivo experiments, yield-contributing and biochemical traits were measured. The parameters included plant height, plant number, total tiller numbers, spike length, number of grains per spike, 1000-grain weight, grain yield per plant (g), and protein percentage, which were studied under irrigated and water-stress conditions. Drought stress significantly reduced seedling and yield-contributed traits. In the case of biochemical parameters (protein and proline), an increase was observed. The results can provide insight into the root trait development of wheat under -0.45 MPa and guide root architecture optimization and quality improvement in wheat. An analysis of correlations found significant correlations between most of the studied traits. According to the results, the cultivars Navid, Sabalan, Azar2, and Zare were identified as drought-tolerant while the cultivars Mihan, D92, and G31 were observed as drought-sensitive.