In the present study, chemical mutagenesis was used to induce mutations in the wheat variety NN-Gandum-1 while gamma rays (γ rays) were used to induce mutations in Punjab-11 (Pb-11). The aim of mutagenesis was to improve resistance to the disease as well as to study function of genes conferring resistance to the disease. A total of 3,634 M5 mutant lines of NN-1 and 3,600 M5 mutant lines of Pb-11, 3,533 M6 lines mutant of NN-1 and 3,483 M6 mutant lines of Pb-11 and 3,502 M7 mutant lines of NN1 and 3,453 M7 mutant lines of Pb-11 were produced and characterized for days to heading (DH), plant height (PH), spike length (SL), tillers per plant (TPP), and resistance to leaf rust (LR) and yellow rust (YR). Across all the measured traits of three mutant generations, ranges of phenotypic values among the mutant lines were significantly larger than the corresponding values of wild type of both populations (0.57–12.52 for NN-1 and 0.62–11.5 standard deviations for Pb-11). Significant correlations of the traits between mutant generations confirmed the genetic basis for these traits. A subset of 239 M7 lines (17 NN-1, 222 Pb-11) were selected for their resistance to LR and YR. These lines also showed phenotypic variations for PH (12 NN-1, 137 Pb-11), DTH (9 NN-1, 14 Pb-11), TPP (6 NN-1, 52 Pb-11) and SL (12 NN-1, 81 Pb-11). For all these traits, mutant lines showed significant improvements as compared to the corresponding wild type. Cumulatively, Pb-11 mutant population exhibited more mutants (26.18%) than the EMS-treated NN-1 population (22.76%), owing to various genetic backgrounds, type of mutagen used and environmental conditions. In the M4 generation, a total of 11 M4 lines (nine absolute resistant and two highly susceptible) and one wild type were selected for NGS-based exome capture assay. A total of 104,779 SNPs were identified that were randomly distributed throughout the wheat sub genomes (A, B and D). Induced mutations in intronic sequences predominated. The highest total number of SNPs detected in this assay were mapped to chr.2B (14,273 SNPs), which contains the highest number of targeted base pairs in the assay. The average mutation density across all regions interrogated was estimated to be one mutation per 20.91 Mb. The highest mutation frequency was found in chr.2D (1/11.7 kb) and the lowest in chr.7D (1/353.4 kb). Out of the detected mutations, 101 SNPs were filtered using analysis criteria aimed to enrich for mutations that may affect gene function. Out of these, one putative SNP detected in xv Lr21 were selected for further analysis. The SNP identified in chimeric allele (Lr21) of a resistant mutant (N1-252) was located in a NBS domain of chr.1BS at 3.4 Mb position. These lines were again screened for resistant to the rust diseases along with their yield response at multiple locations for three consecutive crop years (2014-2016). In these trials, PGMB-15-30 was found the most resistant among all the mutants and showed higher grain yield potential compared to the wild type NN-Gandum-1, Morocco and local variety Galaxy13. In the national wheat disease screening nursery (NWDSN) trials, PGMB-15-30 demonstrated high resistance to LR and YR in three provinces of Pakistan during 2015-2016. Through computational analysis, it was demonstrated that the identified SNP causes a substitution of glutamic acid with alanine, resulting in a predicted altered protein structure. This mutation, therefore, is a candidate for contributing towards resistance phenotype in the mutant line. The newly developed wheat mutant resources can contribute novel alleles which can be used in developing new wheat cultivars as well as in getting insights into various biological circuits of different complex traits in wheat.