Tritordeum is a crop that is gaining interest in the last years, particularly to be used in the production of high quality artisan baked products, mainly bread. This cereal was synthesized by the cross between durum wheat and South-American wild barley (Hordeum chilense) as female parent. Although multiple primary tritordeum were carried out using diverse durum wheat and H. chilense lines, the current tritordeum cultivars derive from a small set of these original materials that could have suffered diversity loss through genetic drift processes. This would justify the search of additional variation in H. chilense to recuperate diversity in tritordeum through new crosses. Here, ten H. chilense lines used in the past for synthesized several primary tritordeum have been characterized for their high-molecular-weight glutenin subunits, detecting up to ten different alleles. The results suggested that the nucleotide sequences of these alleles can be grouped in two sets, corresponding with previous biotypes defined for this species. The sequences within each set showed important differences among them; some of the amino acid changes could have influence on the tritordeum bread-making quality. Consequently, its introgression could increase the range of gluten strength in tritordeum, expanding its potential uses within of the food industry.