Barley grain protein is one of the important components to determine the nutritive value for the whole grain diet and feed, and to have a significant influence on the quality of malt and beer. In particular, low protein content is required for high malt quality, and is directly related to N fertilization. Since genes/QTL highly associated with high or low protein phenotypes were introduced into barley breeding populations, we have conducted genetic analysis to identify the related QTLs and their genetic effects on the phenotype using DNA markers. The research was conducted by testing F₅-derived 100 lines from the cross of the six-rowed hulled barley cultivar cv. Karl showing the consistent low grain protein content and the two-rowed malt barley cultivar originating from Korea cv. Jinyangbori for the agronomic traits and grain protein level at one location over 2yrs and two locations in 1yr. Each row plot was N-fertilized at the heading stage of individual lines with a rate of 150% of basal N. These lines were also evaluated for 88 DNA markers to map QTLs associated with grain protein content. The grain protein content of recombinant lines ranged between 10.4-16.8% with a significant genotypic variation (P < 0.05). The effects of interaction between genotype and environment, and heritability estimates will be analyzed on the variation of grain protein content. For QTL mapping, nine linkage groups were constructed with 72 DNA markers. By single point analysis using ANOVA, at least three QTLs were mapped on chromosomes 2H, 6H and 7H explaining 7.1-40.4% of the variability for grain protein content. Major QTL greatly associated with low protein content might be located around the SSR marker HVM14 on chromosome 6H. More than 30% of the total phenotypic variance of protein level could be explained by this region.