Growing crops with improved N use efficiency (NUE) could decrease fertilizer inputs and the negative environmental effects of N overuse. We measured nine agronomic and yield component traits under N-limiting and nonlimiting treatments for 250 two-row and 250 six-row barley (Hordeum vulgare L.) breeding lines. We calculated four stress indices to approximate NUE. To identify quantitative trait loci (QTLs), we conducted association mapping (AM) for these phenotypic data with 3072 single nucleotide polymorphism markers. All traits and two stress indices—the geometric mean and the stress tolerance index—exhibited significant genetic variation within the two-row and six-row panels. Of the 25 QTLs identified in the two-row panel and 31 identified in the six-row panel, only four were common to both. Four of the QTLs detected in the two-row panel and 10 detected in the six-row panel were identified under the N-limiting treatment or for a stress index, but not under the nonlimiting treatment, signaling that they may be associated with NUE. We detected a QTL for grain protein concentration (GPC) on chromosome 6H that has been mapped previously in barley and is collinear with the well-characterized Gpc-B1 locus in wheat. Groups of lines defined by marker haplotypes at this locus exhibited significant differences in GPC but not in grain yield. These results support that breeding strategies for improving NUE include crossing between two-and six-row parents with complementary alleles, phenotypic selection based on stress indices, marker-assisted selection for desirable alleles, and genomic selection to capture small-effect loci.