[Objective] Jasmonate-amido synthetase gene is a crucial component of the jasmonic acid (JA) signaling pathway in plants. This study aimed to explore the protein structure and expression pattern of HvnJAR1 and HvnJAR2, providing insights into the JA signaling pathway in hulless barley. [Methods] Bioinformatics analysis and qPCR were used for analyzing the protein structure and expression patterns of HvnJAR1 and HvnJAR2. [Results] HvnJAR1 and HvnJAR2 promoter regions contained elements associated with phytohormones and stress responses. Both HvnJAR1 and HvnJAR2 has no signal peptide and transmembrane, but had a highly conserved GH3 domain. HvnJAR1 and HvnJAR2 were localized in the chloroplast and most closely related to BdJAR1 and BdJAR2. HvnJAR1 and HvnJAR2 had similar secondary, tertiary, cavity and channel structures. HvnJAR1 exhibited stronger binding ability for the HvnGST dimer compared with HvnJAR2. HvnJAR1 and HvnJAR2 were highly expressed in seeds, anthers, and tillers compared with other tissues, and their expression was induced by low temperature, drought, NaCl, MeJA, and ABA. [Conclusion] HvnJAR1 and HvnJAR2 may play significant roles in regulating growth and responding to adversity in hulless barley.