Salinity is a major constraint for plant growth, development and yield worldwide. Evaluation of a large number of germplasms in salt-stressed environments may help identify superior salt-tolerant genotypes. The present study dissects the genetic diversity of 33 pearl millet genotypes (landraces and inbred lines) for salinity tolerance through in vitro screening at the seedling stage. Our results revealed a significant reduction in total biomass and shoot growth of the salt-sensitive genotypes upon exposure to 150 mM NaCl, in contrast to the tolerant genotypes showing better growth characteristics. A significant differential effect of salt treatment on morphological traits was observed by analysis of variance (ANOVA), confirming substantial genetic diversity among all genotypes for salt tolerance. The genotypes were clustered into three groups based on multiple stress indices. The genotypes were also evaluated using principal component analysis (PCA) to identify the key contributing traits for stress tolerance. Based on these results, a total of four contrasting genotypes were selected for further biochemical and molecular analysis. Physiological studies confirmed that salt tolerance might be due to the higher content of osmolytes and the activity of antioxidant enzymes. Similarly, gene expression profiling of catalase (CAT), glutamate dehydrogenase (GDH), glutathione reductase (GR), and nitrate reductase (NR) revealed a profound increase in NR and GDH transcript levels in the tolerant genotypes, suggesting their major role as reactive oxygen species (ROS) scavengers under salinity. The overall findings of this study could be utilized further for candidate gene mining through "omics" approaches, aiming toward development of salinity resilient crop plants.

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Salinity stress poses a significant risk to plant development and agricultural yield. Therefore, elucidation of stress‐response mechanisms has become essential to identify salt‐tolerance genes in plants. In the present study, two genotypes of pearl millet (Pennisetum glaucum L.) with contrasting tolerance for salinity exhibited differential morpho‐physiological and proteomic responses under 150 mM NaCl. The genotype IC 325825 was shown to withstand the stress better than IP 17224. The salt‐tolerance potential of IC 325825 was associated with its ability to maintain intracellular osmotic, ionic, and redox homeostasis and membrane integrity under stress. The IC 325825 genotype exhibited a higher abundance of C4 photosynthesis enzymes, efficient enzymatic and non‐enzymatic antioxidant system, and lower Na+/K+ ratio compared with IP 17224. Comparative proteomics analysis revealed greater metabolic perturbation in IP 17224 under salinity, in contrast to IC 325825 that harbored pro‐active stress‐responsive machinery, allowing its survival and better adaptability under salt stress. The differentially abundant proteins were in silico characterized for their functions, subcellular‐localization, associated pathways, and protein–protein interaction. These proteins were mainly involved in photosynthesis/response to light stimulus, carbohydrate and energy metabolism, and stress responses. Proteomics data were validated through expression profiling of the selected genes, revealing a poor correlation between protein abundance and their relative transcript levels. This study has provided novel insights into salt adaptive mechanisms in P. glaucum, demonstrating the power of proteomics‐based approaches. The critical proteins identified in the present study could be further explored as potential objects for engineering stress tolerance in salt‐sensitive major crops.