Soybean is a major crop in the world and an essential source for minerals, including iron (Fe) and zin (Zn). Deficiency of Fe and Zn in soil and soybean plants result in yield loss and poor seed nutritional qualities. Information on genomic regions and candidate genes controlling seed Fe and Zn accumulation in soybean seed is limited. Therefore, The objective of this research was to identify genetic regions, known as quantitative trait loci (QTL), and candidate genes that control the accumulation of Fe and Zn in soybean mature seeds. A ‘Forrest’ by ‘Williams 82’ (F × W82) recombinant inbred line (RIL) population (n = 306) was used and genotyped using a total of 5405 single nucleotides polymorphism (SNP) markers using Infinium SNP6K BeadChips. A two-year experiment was conducted across two environments: North Carolina in 2018 (NC) and Illinois in 2020 (IL). Only QTL with LOD scores ≥ 2.5, as identified by the composite interval mapping (CIM) method, are reported here. In total, 6 QTL were identified for seed Fe; specifically, 3 QTL (qFe-01-[NC-2018], qFe-02-[NC-2018], and qFe-03-[NC-2018]) were located on chromosomes 1, 2, and 6, respectively, in the NC environment, and 3 QTL (qFe-01-[IL-2020], qFe-02-[IL-2020], and qFe-03-[IL-2020]) were positioned on chromosomes 1, 2, and 12, respectively, in the IL environment. A total of 6 QTL associated with seed Zn were also identified; 4 QTL (qZn-01-[NC-2018]; qZn-02-[NC-2018]; qZn-03-[NC-2018]; and qZn-04-[NC-2018]), respectively on Chr 2, 3, 7, and 19 in NC; and 2 QTL (qZn-01-[IL-2020] and qZn-02-[IL-2020]), respectively, on Chr 5 and 8 in IL. Several functional genes encode Fe- and Zn-proteins, transcription factors, proteins-zinc finger motifs (involved in DNA binding and transcriptional regulation; crosstalk between the regulatory pathways of Zn and Fe transporters) were identified and located within the QTL interval. To our knowledge, and based on the literature available, the QTL identified here on Chr 2 and Chr 6 are novel and were not previously identified. This current research provides a new knowledge of the genetic basis of seed Fe and Zn and the markers associated with QTL. The QTL identified here will contribute to efficient marker assisted selection for higher Fe and Zn content in soybean seeds. The candidate genes and metal-responsive transcription factors may coordinate the expression of both Zn and Fe transporters in response to changes in metal availability, providing new knowledge on minerals uptake and transport mechanisms, allowing for possible genetic engineering application.