Wheat is the major nutrient source worldwide and has a crucial place in agriculture and economy of Pakistan. Characterization of wheat genetic resource is imperative for breeders in developing databases helpful for parental selections as presence of variance has primary significance with respect to plant breeding. In this view, 77 wheat genotypes collected from different agro-climatic zones of country were characterized for seed biochemical and nutritional quality traits, distribution of glutenin subunits, allelic variations in Xuhw89 locus for grain protein content and yield parameters followed by cluster, principal component (PCA) and correlation analysis. Seed biochemical analysis, revealed a significant variation (p < 0.05) in genotypes for nutritional potential. Genotypic sources were identified for better antioxidant potential i.e. Pavon (APX), Pasban-90 (CAT), IQBAL-2000 (POD), Manthar-2003 (SOD), Bhakkar-2000 (TPC), hydrolytic enzymes i.e. SH-2002 (amylase and ascorbic acid), Dharabi 2011 (esterase), NR-234 (protease), grain protein i.e. Faisalabad-2008 (salt soluble protein), TC-4928 (albumins), MEXI PAK (globulins) and sugars i.e. Saleem 2000, Punjab-96 (sugars and soluble protein). Dharabi-2011 with lowest amylase, MEKI-PAK with lowest reducing sugars and higher seed protien and AARI-2011 with lowest gluten have potential for health implications. Correlations in seed nutritional parameters were identified while genotypes were clustered in four groups with highest distance between cluster- II and III. However, genotypes grouped into five clusters based on yield related traits having maximum distance in cluster-II and IV with spike length as principal component of varibility. Based on divergence in single kernel characteristics, genotypes with highest protein (GA2002, 16.5%), moisture (Marvi, 16.5%), starch (9244, 11%), zeleny (AARI-2011, 54.1%), gluten (2006, 44%), kernel weight (TC-4928, 41.6±9.5mg), kernel diameter (Sassui, 2.91±0.32mm), hardness index (Punjab 201191±39%) were identified and grouped in five clusters. Kernel weight, diameter, moisture and starch content were principal components of variability. SDS-PAGE revealed 30 allelic combinations for high molecular weight glutenin subunits (HMW-GS) at Glu-1 loci involved in bread making quality. 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 were detected in 33 and 32 genotypes at Glu-B1 as well as Glu-D1 locus respectively. Few rare alleles were also detected. The quality scores ranged from 4-10, while highest quality score (10) was more frequent (36.36%). Analysis of microsatellite (functional SSR marker) Xuhw89 locus for grain protein content showed that 42% genotypes carry 126+130-bp allele with relatively higher salt soluble proteins and globulin while remaining possessed a 126-bp allele with higher total soluble protein and albumin. In conclusion, revealed divergence in seed biochemical and quality traits, allelic distribution for HMW-GS and Xuhw89 locus for grain protein content and yield parameters in Pakistani wheat germplasm followed by detailed cluster, PCA and association analysis will help breeders in selecting genotypes with desirable traits and designing the breeding program for crop improvement.