Polyamines (PAs) are small molecules that exist in all living organisms. They play a crucial role in various physiological processes in plants, including growth, development and responses to stress. Cold stress is one of the major abiotic stresses that has a negative effect on the growth, development and productivity of plants. In the present study, exogenous PAs (Spd, Put and distilled water as control) were efficiently used in mitigating of stress effects in two contrasting wheats cultivars (‘Mihan’ and ‘Rakhshan’) grown under cold conditions. Shoot and root dry weight, chlorophyll, carotenoid, proline contents, catalase and ascorbate peroxidase activities were improved by PA application (10 mg L−1 foliar spray) in the cold-stressed plants. The gene expression analysis with RT-qPCR showed transcriptional variation of selected genes in response to cold condition and PAs. Under cold condition the expression levels of genes responsible for the synthesis (TaADC, TaSAMDC, TaSPDS) and catabolism (TaPAO11–7B TaPAO11–7D) increased in both cultivars, but at higher level in the tolerant one. These results reveal a significant role of PAs under cold acclimation in wheat leaves.