GS1andGS2genes encode, respectively, the main cytosolic and the plastidic isoforms of glutamine synthetase (GS). In the present study, the wheatGS1andGS2homoeogenes located in the A, B and D genome chromosomes have been sequenced in a group of 15 bread wheat varieties including landraces, old commercial varieties and modern cultivars. Phenotypic characterization by multi-environment field trials detected significant effects of specific GS homoeogenes on three of the seven agronomic and grain quality traits analyzed. Based on the gene sequence polymorphisms found, biallelic molecular markers that could facilitate marker-assisted breeding were developed for genesGS1A,GS2AandGS2D. The remaining genes encoding main wheat GS were excluded because of being monomorphic (GS1D) or too polymorphic (GS1BandGS2B) in the sequencing panel varieties. A collection of 187 Spanish bread wheat landraces was genotyped for these gene-based molecular markers. Data analyses conducted with phenotypic records reported for this germplasm collection in López-Fernández et al. (Plants-Basel 10: 620,2021) have revealed the beneficial influence of some individual alleles on thousand-kernel weight (TKW), kernels per spike (KS) and grain protein content. Furthermore, genetic interactions betweenGS1A, a cytosolic GS isoform coding gene, andGS2AorGS2D, plastidic GS enzyme coding genes, were found to affect TKW and KS. The finding that some alleles at one locus may mask the effect of positive alleles at hypostatic GS loci should be kept in mind if gene pyramiding strategies are attempted for the improvement of N-use efficiency-related traits.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11032-022-01354-0.