Abstract: Hulless barley is the principal staple crop in the Qinghai-Tibet Plateau region. Due to its predominant cultivation in high-altitude alpine zones above 2700 meters, its growth and development are subject to multidimensional constraints imposed by low-temperature stress. MYB transcription factors are involved in abiotic stress responses in a variety of crops, but no related studies have been reported in highland barley. In this study, the function of HvMYB15 gene in response to low temperature stress was explored by bioinformatics analysis, gene expression analysis and heterologous overexpression function verification in Arabidopsis thaliana. The results showed that the full-length coding region of HvMYB15 gene was 867 bp, encoding 289 amino acids, and its promoter region contained multiple cis-acting elements related to stress response. qRT-PCR showed that HvMYB15 was expressed at higher levels in roots, followed by leaves. Stress-induced expression analysis showed that low temperature stress could significantly induce the up-regulated expression of HvMYB15 gene. Subcellular localization studies and transcriptional activation assays demonstrated that HvMYB15 exhibits nucleus localization and possesses transcriptional self-activation properties. The functional verification of low temperature stress showed that the seedling survival rate of HvMYB15 overexpression lines was significantly higher than that of the wild type (WT), the ion leakage rate, hydrogen peroxide, and malondialdehyde content were significantly lower than those of the WT, while the superoxide dismutase activity and proline content were significantly higher than those of the WT. In addition, the expression levels of genes related to low temperature stress response in HvMYB15 overexpression lines were significantly up-regulated compared with WT after low temperature stress treatment. In summary, this study shows that the HvMYB15 gene of highland barley has a positive regulatory effect on low temperature stress. The results of this study lay a theoretical foundation for further analysis of the molecular mechanism of low temperature tolerance response and genetic breeding improvement of hulless barley.