The gluten strength and the composition of high- and low-molecular-weight glutenin subunits (HMWGSs and LMWGSs) of fifty-one durum wheat genotypes were evaluated using sodium dodecyl sulfate (SDS) sedimentation testing and SDS polyacrylamide gel electrophoresis (SDS-PAGE). This study examined the allelic variability and the composition of HMWGSs and LMWGSs in T. durum wheat genotypes. SDS-PAGE was proven to be a successful method for identifying HMWGS and LMWGS alleles and their importance in determining the dough quality. The evaluated durum wheat genotypes with HMWGS alleles 7+8, 7+9, 13+16, and 17+18 were highly correlated with improved dough strength. The genotypes containing the LMW-2 allele displayed stronger gluten than those with the LMW-1 allele. The comparative in silico analysis indicated that Glu-A1, Glu-B1, and Glu-B3 possessed a typical primary structure. The study also revealed that the lower content of glutamine, proline, glycine, and tyrosineand the higher content of serine and valine in the Glu-A1 and Glu-B1 glutenin subunits, and the higher cysteine residues in Glu-B1 and lower arginine, isoleucine, and leucine in the Glu-B3 glutenin, are associated with the suitability of durum wheat for pasta making and the suitability of bread wheat with good bread-making quality. The phylogeny analysis reported that both Glu-B1 and Glu-B3 had a closer evolutionary relationship in bread and durum wheat, while the Glu-A1 was highly distinct. The results of the current research may help breeders to manage the quality of durum wheat genotypes by exploiting the allelic variation in glutenin. Computational analysis showed the presence of higher proportions of glutamine, glycine, proline, serine, and tyrosine than the other residues in both HMWGSs and LMWGSs. Thus, durum wheat genotype selection according to the presence of a few protein components effectively distinguishes the strongest from the weakest types of gluten.