Nitrogen (N) stress seriously constrains barley (Hordeum vulgareL.) production globally by influencing its growth and development. In this study, we used a recombinant inbred line (RIL) population of 121 crosses between the variety Baudin and the wild barley accession CN4027 to detect QTL for 27 traits at the seedling stage in hydroponic culture trials and 12 traits at the maturity stage in field trials both under two N treatments, aiming to uncover favorable alleles for N tolerance in wild barley. In total, eight stable QTL and seven QTL clusters were detected. Among them, the stable QTLQtgw.sau-2Hlocated in a 0.46 cM interval on the chromosome arm 2HL was a novel QTL specific for low N. Notably, Clusters C4 and C7 contained QTL for traits at both the seedling and maturity stages. In addition, four stable QTLs in Cluster C4 were identified. Furthermore, a gene (HORVU2Hr1G080990.1) related to grain protein in the interval ofQtgw.sau-2Hwas predicted. Correlation analysis and QTL mapping showed that different N treatments significantly affected agronomic and physiological traits at the seedling and maturity stages. These results provide valuable information for understanding N tolerance as well as breeding and utilizing the loci of interest in barley.