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Wheat is one of the most important cereal crops in the world. Cold stress is a major constraint in production of wheat grown in cold climate regions. In this study, we conducted a comprehensive assessment of cold stress tolerance in wheat genotypes through field screening, cell membrane stability through electrolyte leakage assay and biochemical profiling. A core set comprising 4560 genotypes was evaluated for two years (2021–2022), revealing substantial genetic variation for cold stress tolerance. Most genotypes exhibited moderate tolerance, while a smaller proportion showed susceptibility to cold stress. Based on the cold screening data in the field, a mini‐core set of 350 genotypes was selected for membrane stability analysis using electrical conductivity assays. Significant differences were observed in membrane stability among the genotypes, indicating the presence of genetic variation for this trait. Furthermore, a mini‐core set was narrowed down to 50 diverse candidate genotypes that were subsequently profiled for various biochemicals, including reactive oxygen species (ROS) like lipid peroxidation (MDA) and hydrogen peroxide (H202), osmoprotectant (proline) and enzymatic antioxidants including ascorbate peroxidase (APX), superoxide dismutase (SOD), guaiacol peroxidase (GPX), and catalase (CAT). Correlation analysis of the biochemicals revealed negative associations between antioxidants and reactive oxygen species (ROS), highlighting their role in mitigating oxidative damage under cold stress. This study enhances our understanding of the physiological and biochemical mechanisms underlying cold stress tolerance in wheat. The identified genotypes with superior cold stress tolerance can serve as valuable genetic resources for wheat breeding. [ABSTRACT FROM AUTHOR]