Wheat is a staple diet due to its breadmaking quality, which is governed by seed storage proteins. This research evaluated genetic variability in morphological attributes and profiled gluten proteins in wheat genotypes. Sixty wheat varieties from Pakistan were assessed for agronomic traits, while the diversity of endosperm storage proteins in sixty-six genotypes was evaluated using SDS-PAGE. Moderate heritability for plant height (0.60) and flag leaf area (0.54) was noted in Khyber Pakhtunkhwa, while Punjab showed moderate to high heritability for heading days (0.67), plant height (0.64), flag leaf area (0.81), and flowering days (0.77). Sindh exhibited high heritability for spike length (0.88). A total of 61 alleles were found in 27 genotypes in Punjab, 49 in 15 genotypes in Sindh, and 48 in 24 genotypes in KP. Cluster analysis revealed Bhittai as the most diverse genotype in Sindh, DN lines in KP, and AS-02 in Punjab. These genotypes show significant diversity in gluten proteins, which are crucial for grain quality traits. The study concludes that wheat genotypes have sufficient variation in morpho-agronomic traits and protein profiling, which is useful for developing high-quality genotypes. Future research should explore the genetic basis of gluten protein diversity and its relationship with wheat quality traits using advanced genomic tools.