Glutamine synthetase (GS2) is a key enzyme in plant nitrogen metabolism responsible of the first step of ammonium assimilation and transformation into glutamine (an essential compound in the amino acid-biosynthetic pathway). The goal of the present study was to isolate and characterize GS2 genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes. Here, we report the isolation of the complete glutamine synthetase gene sequences and their localization on the two homeologous chromosome 2A and 2B in durum wheat cvs. Ciccio and Svevo characterized by a different grain protein content. GS2-A2 located on 2A chromosome is comprised of 13 exons separated by 12 introns, and the allele sequence in the two cultivars were different for an insertion of 5 bp located in the third exon in the cv. Ciccio. The GS2-B2 has the same intron/exon structure, but the two cultivars differ for the insertion of a 33-bp sequence located in the second intron of the cv. Svevo. Specific primers were designed in the polymorphic region and amplified in a recombinant inbred line mapping population. The study localized GS genes (GS2-A2, GS2-B2 GSe, GSr) on chromosomes 2A, 2B, 4A, and 4B, where four significant QTLs for GPC were also located.