Grain Protein Content (GPC) is an important determinant of grain quality in many crops, including barley and wheat. Recently, the map-based cloning of Gpc-B1, a wheat GPC quantitative trait locus (QTL), revealed a NAC transcription factor (TtNAM-B1) that was associated with increased grain protein, zinc, and iron content. In barley, a QTL for GPC was identified in a segregating population developed from a cross between 'Karl' (low GPC) and 'Lewis' (average GPC). This QTL was mapped near marker hvm74 on chromosome 6H and was suggested as a potential orthologue for Gpc-B1 on chromosome arm 6BS. In the current study, wheat genes that were previously mapped within a 0.8 cM segment spanning the TtNAM-B1 gene were converted into barley molecular markers. These new markers, together with the barley TtNAM-B1 orthologous gene (designated HvNAM-1 hereafter) were mapped on a 0.7 cM interval encompassing the peak of the barley QTL for GPC on chromosome arm 6HS. Sequence comparison of HvNAM-1 parental alleles showed two single nucleotide polymorphisms (SNPs) that result in two amino acid differences. Analysis of the allelic variation in a wild and cultivated barley collection revealed that the Karl haplotype was present only in nine out of 147 tested accessions. The colinearity between the wheat and barley GPC regions and the low frequency of the HvNAM-1 haplotype associated with low GPC suggest that the barley NAC transcription factor is responsible for the GPC QTL on barley chromosome 6H.