The aim of this work was to evaluate the main quality traits in the parental lines of wheat segregating populations to identify the best for subsequent genetic mapping of the traits. Significant differences (p < 0.001) among wheat genotypes were observed. Many of the examined crosses appeared to be suitable for the purpose, showing differences among parental lines as high as 4.6% for protein content, 6.4% for gluten content, 69 for gluten index, 50 mL for sodium dodecyl sulphate sedimentation volume, and 33.9 g for thousand-kernel weight, whereas differences accounting for 4.8, 2.4, and 7.3 were observed for yellow index, red index and brown index, respectively. The results pointed out that for studying at the same time the quantitative and qualitative features of gluten, the wheat populations derived from Latino x MG29896 and Saragolla x 02-5B-318 could be particularly appropriate. In addition, the latter cross was suitable to deepen the knowledge of yellow index regulation.