Autumn cultivation of desi-type chickpea in cold regions requires the introduction of cultivars tolerant to freezing stress. Hence, this study aimed to investigate the physiological response of desi-type chickpea germplasm during cold acclimation and its relationship with freezing tolerance (−12 °C) under controlled conditions. A genotype with a higher potential for accumulation of anthocyanins, proline, and DPPH (free radical scavenging activity) in cold acclimation duration was identified as a promising genotype with lower electrolyte leakage and 100% survival. In the germplasm, 81% of the genotypes kept 100% survival. No significant correlation was observed between the content of metabolites in cold acclimation duration and the survival percentage of genotypes after freezing stress. While the correlation between survival percentage and electrolyte leakage was negative and significant, except for one genotype, the other genotypes with the lowest EL had 100% survival. Principal component analysis indicated the first component screened genotypes with higher content of photosynthetic pigments and the second component screened genotypes with more survival and cell membrane stability after freezing stress. Morphological parameters explain the freezing tolerance better than metabolites. Based on the results of this experiment, it is recommended to evaluate the content of photosynthetic pigments before freezing stress and assess the stability of cell membranes after freezing stress to measure the tolerance to freezing stress in genetic engineering and breeding programs.

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