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Understanding how plants respond to salinity stress is essential for developing tolerant genotypes, to keep human food secure since it is threaten by climate changes and increasing population worldwide. Barley (Hordeum vulgare) is a crop that possesses various salinity tolerance mechanisms that remain to be explored. In this study, data from an RNA-Seq experiment in barley was analyzed to identify changes in genome activities as well as differentially expressed genes (DEGs) in response to salinity stress. A gene network was predicted among identified DEGs and was subjected to network topology analysis, which resulted in the prediction of a hub gene, namely low expression of osmotically responsive gene 2 (LOS2). LOS2 and its two hierarchical downstream genes, salt-tolerant zinc finger (ZAT10) and ascorbate peroxidase 1 (APX1), were used in a genome-wide association (GWA) survey to confirm their importance. A field experiment was conducted to recognize susceptible and tolerant genotypes among 10 different barley genotypes based on the principle component analysis (PCA) of stress-related indices. In a separate salinity experiment, two of the genotypes were assessed to assign their physiological and biochemical responses as well as to identify expression profiles of LOS2, ZAT10, and APX1. From the results, the activity of the barley genome was significantly altered toward response to stress. In total, 5692 DEGs were identified and the gene network derived from these genes contained 131 nodes and 257 edges. The identified genotypes clearly showed the difference in water status, osmolyte accumulation, cell membrane damages, and ion homeostasis as well as in expression profiles for studied genes during salinity stress. Our results suggest that LOS2 along with the ZAT10 and APX1 genes may serve as an important part of barley salinity stress tolerance pathways. To our knowledge, this is the first report on the role(s) of LOS2 in barley salinity stress tolerance in a gene network system.