Hordeum chilense is an excellent genetic resource for wheat breeding due to its potential to improve breadmaking quality and nutritional value and provide resistance to some biotic and abiotic stresses. Hexaploid wheat lines carrying chromosome 7H(ch) introgressions, namely, chromosome additions of the whole chromosome 7H(ch) or the 7H(ch)α or the 7H(ch)β chromosome arms, and chromosome substitutions of the homeologous chromosomes 7A, 7B, or 7D by chromosome 7H(ch) were compared by 2D-PAGE analysis to study the effect of these alien introgressions on the wheat endosperm proteome. The addition of the 7H(ch)α chromosome arm did not alter the profile of most glutenins and gliadins, but showed higher quantities of puroindolines and lower xylanase inhibitors, which might improve also resistance to plant pathogens. On the other hand, (7A)7H(ch) or (7D)7H(ch) substitution lines showed enhanced avenin-like b proteins and triticin levels but reduced puroindolines, which could be desirable to improve dough properties and nutritional value and increase kernel hardness in wheat.