The WRKY transcription factors belong to an evolutionarily conserved superprotein family predominantly present in the plant kingdom. WRKY proteins of barley are not yet fully annotated and most of them are not functionally characterized. We performed a genome-wide identification of WRKY members based on the recently accessible barley draft genome sequence and full-length cDNA datasets. As a result, 34 novel putative proteins have been identified which extend the existing list for barley WRKYs to 94. Phylogenetic analysis of the WRKY domains allowed ranking into three groups (I, II, III), with an expansion in group III in monocots. Two members of subgroup IIa, the wound and pathogen-inducible HvWRKY1 and HvWRKY2, are known as negative defense regulators. Here, we demonstrate that both transcription factors repress the activity of the powdery mildew-induced promoter of HvGER4c, a germin-like defense-related protein. The repression did not require the negative defense regulator MLO nor was it affected by the presence of the R protein MLA12. Moreover, the expression of the Arabidopsis ortholog AtWRKY40 in barley compromised basal resistance to powdery mildew, providing evidence for functional conservation of sequence-related WRKY proteins across monocots and dicots.