AbstractThe popularity of wheat flour tortillas is spreading throughout the world and has become one of the highest‐selling bread products. The USDA Southern Regional Performance Nursery (SRPN) serves as the largest wheat‐growing region in the USA, but the tortilla quality of germplasm in SRPN has remained unknown. This study investigated the kernel and flour quality traits, dough rheological properties, and tortilla quality parameters, and glutenin composition of 131 wheat lines from 1995 to 2007 in SRPN. The high molecular weight glutenin subunits (HMW‐GS) composition and wheat–rye 1RS translocation status of these wheat lines were also detected. A wide variation of wheat and tortilla quality traits was observed among these wheat lines. Association analysis showed that increased protein content and gluten strength significantly decreased the tortilla diameter and improved the tortilla shelf life in terms of 2‐D extensibility parameters (P < .001). The group of wheat lines with acceptable tortilla quality (both diameter >165 mm and day 16 flexibility score >3.0) had medium protein content and dough strength. Compared to wheat lines with 5 + 10 and non‐1RS translocation, those with 2 + 12 and 1RS translocation generally showed lower dough strength and shelf life, and higher tortilla diameter and specific volume (P < 0.01). TAM 302, HBG0358, CO99W192, and CO99534 showed premium tortilla quality with a diameter ≥180 mm and a day 16 flexibility score ≥4.00, which could be used as parents in tortilla wheat breeding program.