Wheat is one of the most important domesticated crops grown around the world. Wheat seed storage proteins represent an important source of energy in wheat, being involved in determination of bread making quality. Seed storage proteins are traditionally classified as gliadins and glutenins. Glutenins are divided into low molecular weight glutenin subunits (LMW-GS), 30-50KDa and high molecular weight glutenin subunits (HMW-GS), 80-120KDa. HMW-GSs are encoded at genes Glu-A1, Glu-B1 and Glu-D1. Although HMW-GSs only occupy around 10% of wheat seed storage proteins, they play a major role in bread making quality. A single hexaploid variety could contain 6 HMW-GSs. Protein profiling is the most effective tool for studying the genetic diversity of HMW-GS in wheat varieties.The objective of this study was to perform the protein profiling of wheat HMW-GSs of 21 Pakistan’s wheat varieties. Different allelic combinations of Glu-1 loci were identified. At Glu-A1 locus,2*, at Glu-B1 locus,17+18 and 7+8 and Glu-D1 locus,2+12 alleles were dominant in 21 experimental wheat varieties.