Seed vitality is a key factor for successful germination of seeds and successful root establishment of crops. However, a cold environment can severely hinder the germination of soybean seeds, resulting in a significant decrease in yield. In this study, the cold tolerance of 205 chromosome segment substitution lines (CSSL) during the germination process was evaluated. CSSL_R22 exhibited higher seed vitality under low-temperature conditions. Five quantitative trait loci (QTL) related to cold tolerance during the germination stage were detected. By combining the QTL analysis results with transcriptome data, we determined that GmKAN1 (Glyma.20G108600) is an important regulatory factor for cold tolerance during seed germination. Preliminary studies have shown that GmKAN1, as a transcriptional repressor of GmARF2 and GmARF8, can regulate auxin synthesis to enhance the tolerance of seeds to cold stress. These results provide valuable insights into the regulatory network related to cold tolerance during soybean seed germination.