Glutenin and gliadin subunits play a key role in flour processing quality by network formation in dough. Wild relatives of crops have served as a pool of genetic variation for decades. In this study, 180 accessions from 12 domesticated and wild relatives of wheat were characterized for the glutenin and gliadin genes with allele-specific molecular markers. A total of 24 alleles were detected for the Glu-A3 and Gli-2A loci, which out of 19 amplified products identified as new alleles. Analysis of molecular variance (AMOVA) indicated that 90 and 65% of the genetic diversity were partitioned within two Aegilops and Triticum genera and their species, respectively. Furthermore, all glutenin and gliadin analyzed loci were polymorphic, indicating large genetic diversity within and between the wild species. Our results revealed that allelic variation of Glu-3A and Gli-As.2 is linked to genomic constitutions so that, Ae. caudata (C genome), Ae. neglecta (UM genome), Ae. umbellulata (U genome) and T. urartu (Au genome) harbor wide variation in the studied subunits. Hence, these species can be used in wheat quality breeding programs.