High molecular weight (HMW-GS) and low molecular weight (LMW-GS) glutenin subunits play a significant role in bread making quality and extensibility, though they signify merely 10% and 40% of the entire seed storage proteins. For the estimation of bread quality on the basis of allelic difference in HMW-GS and LMW-GS at Glu-1 and 3 loci, wheat germplasm (77 genotypes) was collected from diverse agro-climatic regions of Pakistan and characterized by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Thirty distinct allelic arrangements were identified with a sum of thirteen Glu-1 alleles. Maximum frequency of allele 1 was found in twenty-nine genotypes at Glu-A1 locus while high proportion of subunit pairs 13 + 16 and 2 + 12 was detected in 33 and 32 genotypes at Glu-B1 as well as Glu-D1 locus, respectively. Few rare alleles were also separated out. The quality scores ranged from 4–10, however highest quality score of ten was more recurrent (36.36%). A good quality score of 8 and 6 were found in 32.47% as well as 19.48% of genotypes individually. In LMW-GS, seventeen diverse combinations of alleles with aggregate of ten Glu-3 alleles were detected. Glu-A3c and Glu-B3d alleles were observed in 33 (42.85%) genotypes, encoding high sedimentation and protein contents. Hence, this will enable the breeders to utilize both glutenin subunits as biochemical indicator for selecting superior wheat genotypes possessing enhanced bread making quality.