Barley yellow mosaic virus disease, caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), is a major viral disease in barley planting regions of Europe and East Asia countries. In the field, this disease is transmitted by the soil-borne fungus Polymyxa graminis via inoculating the roots. Here, by unlocking the transcriptome datasets, we identified a differentially expressed gene HORVU1Hr1G069640 (referred WRKY55) that was significantly up-regulated after infection with BaMMV. Consistently, by analyzing its expression in a group of naturally-inoculated genotypes, a significantly higher expression of WRKY55 was observed in susceptible relative to resistant genotypes. This gene contains an 870 bp of the coding sequence with a conserved WRKY domain between nucleotides 415’-594’. Phylogenetic analysis revealed an independent branch of this gene, differed to the sequence homologous genes. This gene was highly expressed in roots and young tissues, but rarely expressed in other tissues. By Agrobacterium-mediated transformation into Nicotiana benthamiana, the WRKY55-GFP fusion protein was found in the whole cell. In yeast, no trans-activation activity of WRKY55 was detected and no physical interaction between WRKY55 and either susceptibility factors eIF4E or PDIL5-1 were observed. This work identified a BaMMV-infected up-regulated gene WRKY55, which possibly was involved in infection of BaMMV/BaYMV.