This study explored the natural allelic variation of high molecular weight glutenin subunits (HMW-GS), puroindolines, and wheat bread making (WBM) gene in Mediterranean bread wheat landraces.Within the GLU-1 loci, a rich polymorphism was detected with thirty-five different subunits identified, four at GLU-A1 locus, twenty-one at GLU-B1 locus and ten in GLU-D1 locus, being the predominant subunits combinations 2*, 20x+20y, and 2 + 12, respectively. Null alleles were detected in GLU-B1-1, GLU-B1-2 and GLU-D1-2 genes and rare combinations were observed at low frequencies. The influence of HMW-GS on quality traits was confirmed. Seven different purindoline genotypes were identified in the collection, including two novel mutations at the PINB-D1 gene. The relationship between genotype and grain hardness was not readily apparent, revealing the intricate and multifaceted nature of this trait. The Screening for WBM gene (GWseqVar3 sequence variant) showed presence in few landraces and was correlated with higher loaf volumes (LV) and gluten strength (W).The characterization presented in this work, provides promising targets for further research and breeding efforts to develop wheat varieties with improved end-use quality and adaptability to Mediterranean environments.