Wheat (Triticum aestivum L.) flour properties necessary for optimal tortilla production have not been identified. Transgenic wheats (Triticum aestivum L.) overexpressing high molecular weight glutenin subunit (HMW-GS) 1Dy10 were used to make tortilla and their quality was evaluated. The level of HMW-GS 1Dy10 in flours derived from transgenic wheats was 2.5-5.8-fold greater than in controls. Polymeric proteins in the transgenic samples had a molecular weight distribution shifted toward larger polymers as indicated by increased levels of polymeric proteins present and greater M(w) averages of the largest fractions in the insoluble polymeric proteins. Dough derived from transgenic wheats had greater resistance to extension and lower extensibility than controls. Tortilla quality evaluation revealed that tortillas originated from transgenic wheats had decreased diameter, greater thickness and rupture force, and lower rollability scores and stretchability than controls. The presence of 1RS chromosomal translocations from rye (Secale cereale L.) in transgenic wheat decreased the negative effects of overexpression of HMW-GS 1Dy10, as tortillas made with this flour mostly exhibited quality properties similar to those made from control flour. Results suggested that the negative effects of overexpression of HMW-GS 1Dy10 on tortilla properties were derived from a nonideal gluten matrix formation.