Background: High yielding rice varieties are usually low in grain iron (Fe) and zinc (Zn) content. These two micronutrients are involved in many enzymatic activities, lack of which cause many disorders in human body. Bio-fortification is a cheaper and easier way to improve the content of these nutrients in rice grain.Results: A population panel was prepared representing all the phenotypic classes for grain Fe-Zn content from 485 germplasm lines. The panel was studied for genetic diversity, population structure and association mapping of grain Fe-Zn content in the milled rice. The population showed linkage disequilibrium showing deviation of Hardy-Weinberg’s expectation for Fe-Zn content in rice. Population structure at K = 3 categorized the panel population into distinct sub-populations corroborating with their grain Fe-Zn content. STRUCTURE analysis revealed a common primary ancestor for each sub-population. Novel quantitative trait loci (QTLs) namelyqFe3.3andqFe7.3for grain Fe andqZn2.2,qZn8.3andqZn12.3for Zn content were detected using association mapping. Four QTLs, namelyqFe3.3,qFe7.3,qFe8.1andqFe12.2for grain Fe content were detected to be co-localized withqZn3.1,qZn7,qZn8.3andqZn12.3QTLs controlling grain Zn content, respectively. Additionally, some Fe-Zn controlling QTLs were co-localized with the yield component QTLs,qTBGW,OsSPL14andqPN. The QTLsqFe1.1,qFe3.1,qFe5.1, qFe7.1, qFe8.1, qZn6, qZn7 and gRMm9–1for grain Fe-Zn content reported in earlier studies were validated in this study.Conclusion: Novel QTLs,qFe3.3andqFe7.3for grain Fe andqZn2.2,qZn8.3andqZn12.3for Zn content were detected for these two traits. Four Fe-Zn controlling QTLs and few yield component QTLs were detected to be co-localized. The QTLs,qFe1.1,qFe3.1,qFe5.1, qFe7.1, qFe8.1, qFe3.3, qFe7.3, qZn6, qZn7, qZn2.2, qZn8.3andqZn12.3will be useful for biofortification of the micronutrients. Simultaneous enhancement of Fe-Zn content may be possible with yield component traits in rice.