Barley (Hordeum vulgare L.) is a crop of vital importance for human and animal food security in Algeria. However, in the high plateau and mountainous regions, its yields are severely affected by low-temperature stress and winter frosts.

To address this limitation, a controlled laboratory experiment was conducted to evaluate the cold tolerance of three local barley genotypes (Saida, Tihert, and Oued el Maleh) and to assess the potential role of salicylic acid (SA)—a phytohormone known for its involvement in abiotic stress tolerance—in enhancing plant resilience. Barley seeds were germinated under low-temperature conditions (0–4 °C) for 10 days. Morphological and physiological parameters, such as germination rate and root length, were measured and statistically analyzed using Statistica software.

Significant genotypic differences were observed. In the absence of SA, the Tihert genotype achieved a germination rate of 65%, compared with 20% for Saida and 5% for Oued el Maleh. The application of SA (0.5 mM) markedly enhanced post-germination growth and resilience in the sensitive genotypes, increasing germination rates to 55% for Saida and 25% for Oued el Maleh, while its effect remained limited in Tihert, which already exhibited high natural tolerance.

Hierarchical clustering analysis based on Euclidean distances confirmed Tihert as the most cold-tolerant genotype. The observed improvement in the sensitive genotypes (Saida and Oued el Maleh) suggests that SA may activate antioxidant defense systems and membrane-stabilizing mechanisms.

These findings highlight the importance of genetic diversity among local barley varieties and demonstrate the potential of salicylic acid as a physiological inducer of cold tolerance. Genotypes such as Tihert represent valuable genetic resources for breeding programs aimed at developing barley varieties better adapted to Algeria’s harsh climatic conditions.