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The WRKY gene family consists of transcription factors that are widely distributed in plants and play a key role in plant growth and development, secondary metabolite synthesis, biotic and abiotic stress responses, and other biological processes. In this study, 86 WRKY proteins were identified from the barley genome database using bioinformatics and were found to be distributed unevenly on seven chromosomes. According to the structure and phylogenetic relationships, the proteins could be classified into three groups and seven subgroups. The multiple sequence alignment results showed that WRKY domains had different conserved sites in different groups or subgroups, and some members had a special heptapeptide motif. Protein and gene structure analysis indicated that there were significant differences between the groups in terms of the distribution of WRKY motifs and the number of introns in barley. Tissue expression pattern analysis demonstrated that the transcription levels of most genes exhibited tissue and growth-stage specificity. In addition, the analysis of cis-elements in the promoter region revealed that almost all HvWRKYs had plant hormone or stress response cis-elements, and there were differences in the numbers between groups. Finally, the transcriptional levels of 15 HvWRKY genes under drought, cadmium, or salt stress were analyzed by quantitative real-time PCR. It was found that most of the gene expression levels responded to one or more abiotic stresses. These results provide a foundation for further analysis of the function of WRKY gene family members in abiotic stress.