Wheat endosperm storage proteins are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 5 glutenin loci were identified from a set of 38 tetraploid wheat germplasm obtained from interspecific crosses between durum wheats (Triticum turgidum L. ssp. durum (Desf.) Husn.) and their relatives (T. dicoccum Schübl. and T. polonicum L.) using SDS-PAGE. At Glu-A1 and Glu-B1, encoding high molecular weight glutenin subunits (HMW-GS), 2 and 4 alleles were observed, respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 3, 5 and 3 alleles were identified at loci Glu-A3, Glu-B3 and Glu-B2, respectively. One new allele was detected at Glu-B3 locus and appeared in nine accessions obtained from five crosses. This allele codes for five subunits (2 + 8 + 9 + 13 + 18), encoded by the Glu-B3b without subunit 16 plus subunits 2 and 18. A total of 38 patterns resulted from the genetic combination of the alleles encoding at the five glutenin loci. This led to a significantly higher Nei coefficient of genetic variation in Glu-1, Glu-3 and Glu-B2 loci (0.54). The germplasm analyzed exhibited allelic variation in HMW and LMW glutenin subunit composition and the variation differed from that of tetraploid wheats of other countries. The presence of high quality alleles in glutenin loci have led the accessions to be considered as an asset in breeding programs aimed for wheat quality.