Low temperatures greatly restrict the development, growth, and productivity of soybeans, with their effects differing across various cultivars. The present work investigated the transcriptome and physiological reactions of two soybean cultivars, namely “KD52” exhibiting cold tolerance and “DS17” displaying cold sensitivity, to cold stress across a precisely defined period. The soybean plants were subjected to cold treatment at 6 °C for durations of 0, 2, 4, and 8 h. A comparative physiological marker study revealed distinct reactions to cold stress in the two cultivars. The findings showed that increased malondialdehyde levels provided evidence of DS17’s heightened vulnerability to lipid peroxidation and membrane degradation. In contrast, the KD52 cultivar exhibited increased activities of antioxidant enzymes, including peroxidase and superoxide dismutase, in response to cold exposure, suggesting a strong antioxidant defense system against oxidative stress. The transcriptomic analysis revealed dynamic responses, mapping 54,532 genes. Within this group, a total of 234 differentially expressed genes (DEGs) were found to be consistently changed at several time intervals, showing unique expression patterns across the two cultivars. Analysis of the association between these important DEGs and the physiological indicators revealed candidate genes that may be involved in controlling oxidative damage and antioxidant defenses. Some key genes showed a progressive rise in expression over time in both cultivars, with a more significant acceleration in KD52, and are probably involved in promoting adaptation processes during extended periods of cold exposure. The identification of improved defense mechanisms in KD52, together with the identification of crucial genes, offers great prospects for enhancing the cold stress resilience of soybean.