In order to explore the physiological and molecular response mechanism of highland barley seedlings to salt stress, ZYM1795, ZYM3130 and ZYM2239 highland barley seedlings were used as materials. The seedlings were treated with 50, 150, and 250 mmol·L^-1 NaCl solution and distilled water (control). The contents of malondialdehyde (MDA), total soluble sugar (SS) and the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in the leaves of highland barley were determined. Transcriptome sequencing was performed on the leaves of the control and 250 mmol·L^-1 NaCl treatment. The results showed that with the increase of salt concentration, the MDA content of salt-tolerant materials increased first and then decreased; the activities of SOD, POD and CAT increased first and then decreased; and the SS content accumulated significantly, indicating that highland barley responded to salt stress by activating antioxidant defense system and osmotic adjustment substances. Through transcriptome analysis, a total of 3 343 differentially expressed genes that may be related to salt tolerance were identified. Through weighted gene co-expression network analysis, yellow modules significantly related to all physiological indicators were screened, and 1 801 potential salt tolerance-related genes were further obtained. GO enrichment analysis showed that these genes were mainly enriched in stress response, antioxidant defense, hormone signal transduction, such as abscisic acid response, enzyme activity regulation and other related pathways. Among them, 20 key genes were screened, including peroxidase gene, trehalose phosphorylase gene, serine/ threonine protein kinase gene, and ABC transporter gene. These genes play an important role in active oxygen scavenging, osmotic regulation, and signal transduction. It is speculated that highland barley seedlings enhance salt tolerance by synergistically regulating physiological response and key gene expression.