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Micronutrient and protein malnutrition is recognized among the major global health issues. Genetic biofortification is a cost-effective and sustainable strategy to tackle malnutrition. Genomic regions governing grain iron concentration (GFeC), grain zinc concentration (GZnC), grain protein content (GPC), and thousand kernel weight (TKW) were investigated in a set of 163 recombinant inbred lines (RILs) derived from a cross between cultivated wheat variety WH542 and a synthetic derivative ( Triticum dicoccon PI94624/ Aegilops tauschii [409]//BCN). The RIL population was genotyped using 100 simple-sequence repeat (SSR) and 736 single nucleotide polymorphism (SNP) markers and phenotyped in six environments. The constructed genetic map had a total genetic length of 7,057 cM. A total of 21 novel quantitative trait loci (QTL) were identified in 13 chromosomes representing all three genomes of wheat. The trait-wise highest number of QTL was identified for GPC (10 QTL), followed by GZnC (six QTL), GFeC (three QTL), and TKW (two QTL). Four novel stable QTL ( QGFe.iari-7D.1, QGFe.iari-7D.2, QGPC.iari-7D.2 , and QTkw.iari-7D ) were identified in two or more environments. Two novel pleiotropic genomic regions falling between Xgwm350-AX-94958668 and Xwmc550-Xgwm350 in chromosome 7D harboring co-localized QTL governing two or more traits were also identified. The identified novel QTL, particularly stable and co-localized QTL, will be validated to estimate their effects on different genetic backgrounds for subsequent use in marker-assisted selection (MAS). Best QTL combinations were identified by the estimation of additive effects of the stable QTL for GFeC, GZnC, and GPC. A total of 11 RILs (eight for GZnC and three for GPC) having favorable QTL combinations identified in this study can be used as potential donors to develop bread wheat varieties with enhanced micronutrients and protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krishnappa, Rathan, Sehgal, Ahlawat, Singh, Singh, Shukla, Jaiswal, Solanki, Singh and Singh.)