INTRODUCTION: Exploring cold-tolerant sorghum germplasm is crucial for improving production in low-temperatures regions. However, the cold tolerance of local sorghum germplasms in the Chishui River Basin, located in the southwestern China, remains poorly characterized.METHODS: We evaluated 71 sorghum germplasms at Maotai Institute from 2022 to 2024 using germination tests, pot trials, and field experiments with a two-factor design. Germination potential, germination percentage, and seedling vigor traits (plumule and radicle length and weight) varied significantly among the 71 sorghum germplasm (P < 0.01). Membership function method identified several highly cold-tolerant accessions (e.g., Nos. 12, 22) and cold-sensitive ones (Nos. 17, 44), establishing 15 °C as an optimal temperature for germination stage cold tolerance assessment.RESULTS: Cluster analysis classified these into four groups: cold-tolerant (11), moderately cold-tolerant (22), moderately cold-sensitive (29), and cold-sensitive (9). Agronomic data collected under two early sowing conditions (severe and mild low-temperature stress) demonstrated that several germplasm accessions, like No. 12, maintained high emergence rates (97% and 100% VS. 100% in conventional sowing) and grain weight per panicle (63.3 g and 53.4 g VS. 45.9 g in conventional sowing) without significant reductions, whereas others, such as Nos. 17 and 48, showed marked decreases (P < 0.01). The superior cold tolerance of accessions Nos. 2, 12, and 22 was confirmed through membership function analysis (D-value > 0.6). A significant positive correlation between comprehensive cold tolerance ratings at both the germination and field stages was observed (r = 0.687, P < 0.05). Cold-tolerant germplasms such as No. 12 exhibited high cold tolerance coefficients for chlorophyll content (CHL: 0.98), relative water content (RWC: 0.99), superoxide dismutase (SOD: 1.77), and peroxidase (POD: 2.03), and low malondialdehyde (MDA: 1.20), indicating enhanced membrane stability and oxidative stress tolerance (P < 0.05). Stepwise regression highlighted a strong correlation (r = 0.976, P < 0.01) between predicted values and field D-values, identifying SOD and POD activities as critical physiological indicators of cold tolerance.CONCLUSION: This study not only identifies valuable cold-tolerant sorghum germplasms but also elucidates their physiological mechanisms, providing essential insights and materials for developing cold-tolerant varieties and resilient cultivation practices in the Chishui River Basin.