A total of 232 accessions of tetraploid species, durum wheat (Triticum turgidum L. ssp. durum Desf., 2n=4x=28, AABB) with a widespread origin of various countries were used in this study. Their high molecular weight glutenin subunit (HMW-GS) composition was identified by Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry (MALDI-TOF-MS). Among all accessions analyzed, 194 were homogeneous for HMW-GS, 38 were heterogeneous, and 62 possessed unusual or new subunits. The results revealed a total of 43 alleles, including 5 at Glu-A1 and 38 at Glu-B1, resulting in 60 different allele combinations. The Glu-B1 locus displayed higher variation compared with Glu-A1. Glu-A1c (55.2%) and Glu-B1aj (17.7%) were the most frequent alleles at Glu-A1 and Glu-B1, respectively. Two allele types (“null” and 1) at the Glu-A1 locus and three allele types (7OE + 8, 14+15, 8) at the Glu-B1 locus appeared to be the common types in the 232 accessions. A total of 23 new alleles represented by unusual subunits were detected at the Glu-A1 and the Glu-B1 locus.