© 2016 Mahjourimajd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Genetic variation has been observed in both protein concentration in wheat grain and total protein content (protein yield). Here we describe the genetic analysis of variation for grain protein in response to nitrogen (N) supply and locate significant genomic regions controlling grain protein components in a spring wheat population. In total, six N use efficiency (NUE) field trials were carried out for the target traits in a sub-population of doubled haploid lines derived from a cross between two Australian varieties, RAC875 and Kukri, in Southern and Western Australia from 2011 to 2013. Twenty-four putative Quantitative Trait Loci (QTL) for protein-related traits were identified at high and low N supply and ten QTL were identified for the response to N for the traits studied. These loci accounted for a significant proportion of the overall effect of N supply. Several of the regions were co-localised with grain yield QTL and are promising targets for further investigation and selection in breeding programs.