Grain Protein Content (GPC) of wheat is significant for enhanced nutritional value and is one of the most important aspects effecting pasta and bread making quality as well. Seventy seven Pakistani wheat varieties and advance lines were analysed to access the allelic distribution at microsatellite Xuhw89 locus using functional SSR marker. Overall, 42% of tested wheat genotypes were found to carry 126 + 130-bp allele while a 126-bp allele was detected in 58% of genotypes. A target band of 126-bp was amplified in all tested genotypes, however, an additional band of 130-bp was also detected along with 126-bp band in 32 genotypes. Genotypes i.e. (Punjab-96) total soluble protein and (MEXI PAK) globulin with 126 + 130-bp allele while (Faisalabad-2008) salt soluble protein and (TC-4928) albumin with allele of 126-bp depicted highest grain protein content. The alleles identification associated with maximum grain protein content in Pakistani wheat germplasm will assist in accelerating the breeding program in future.