In this study we analyzed the performance of three wheat varieties in relation to gluten content under high-altitude growing conditions in the Andes of Ecuador. A field experiment was conducted at 3058 meters above sea level during 2009 using adapted wheat cultivar Cojitambo, cv. Carnavalero, and cv. Sibambe. Transcript accumulations of High Molecular Weight Glutenin Subunits (HMW-GS) genes were also evaluated during grain development using qRT-PCR. We recorded the expression profile of HMW-GS genes during 41 days and showed a coordinated pattern of induction with significant higher levels at 82–86 days. Transcript accumulation of 1Dx5, 1Dy10, 1Bx7, 1Ax1, and 1By9 genes were analyzed in more details during this period. The assay highlighted the specific contribution of 1Bx7, 1Dy10, and 1Dx5 during gluten formation in Ecuadorian wheat varieties. Under Andean highlands conditions, cv. Carnavalero showed the higher values of total agglomerated protein upon hydration and higher levels of expression of particular HMW-GS genes. The data suggest a correlation between wet gluten content and HMW-GS genes expression. Our study contributes to understand gluten formation in wheat endosperm under high-altitude conditions in the Andes.