The widespread use of DNA markers has created new opportunities for plant breeding to protect plants from biotic and abiotic stress. The development of rust-resistant wheat varieties is aided by the use of DNA markers, which are helpful in the fight against rust infections in wheat and are essential for the identification, characterization, and expression of rust resistance genes. It is imperative to combine rust resistance with grain quality features. Genes related to grain quality and rust resistance, such as GPC-B1, HMW-GS, Lr34, Lr19, Lr24, and Yr15, are crucial for preventing rust disease-related declines in grain quality and maintaining wheat productivity. Significant correlations were detected between the goal attributes and the molecular validation of markers associated with rust resistance (leaf and yellow) and grain quality genes in a BILs population of bread wheat. The expression of genes such as GPC-B1, HMW-GS, Lr34, Lr19, Lr24, and Yr15 was validated via the use of molecular markers in the validation of rust resistance and grain quality genes within wheat cultivars. In addition to being present alone and in combination with other rust and quality genes, the GPC-B1 gene was found in 82 BIL populations. However, the HMW-GS gene coexisted with additional genes in 169 BILs population. The Lr34 gene was found in 69 populations and was found in different combinations with Lr34/Yr18. Resistance genes to Lr19, Lr24, and Yr15 were also identified; these combinations included two, three, four, and five genes, respectively. Among the 217 chosen BILs, 23 also exhibited all of the genes GPC-B1, HMW-GS, Lr19, Lr24, Lr34, and Yr15. The confirmed common lines will be used for further development of wheat breeding.