Powdery mildew is an important foliar disease of barley (Hordeum vulgare L.) caused by the biotrophic fungus Blumeria graminis f. sp. hordei (Bgh). The understanding of the resistance mechanism is essential for future resistance breeding. In particular, the identification of race-nonspecific resistance genes is important because of their regarded durability and broad-spectrum activity. We assessed the severity of powdery mildew infection on detached seedling leaves of 267 barley accessions using two poly-virulent isolates and performed a genome-wide association study exploiting 201 of these accessions. Two-hundred and fourteen markers, located on six barley chromosomes are associated with potential race-nonspecific Bgh resistance or susceptibility. Initial steps for the functional validation of four promising candidates were performed based on phenotype and transcription data. Specific candidate alleles were analyzed via transient gene silencing as well as transient overexpression. Microarray data of the four selected candidates indicate differential regulation of the transcription in response to Bgh infection. Based on our results, all four candidate genes seem to be involved in the responses to powdery mildew attack. In particular, the transient overexpression of specific alleles of two candidate genes, a potential arabinogalactan protein and the barley homolog of Arabidopsis thaliana's Light-Response Bric-a-Brac/-Tramtrack/-Broad Complex/-POxvirus and Zinc finger (AtLRB1) or AtLRB2, were top candidates of novel powdery mildew susceptibility genes.