Seeds of soybean [Glycine max (L.) Merr.] accumulate more isoflavones than any tissue of any plant species. In other plant parts, isoflavones are usually released to counteract the effects of various biotic and abiotic stresses. Because of the benefits to the plant and positive implications that consumption may have on human health, increasing isoflavones is a goal of many soybean breeding programs. However, altering isoflavone levels through marker-assisted selection (MAS) has been impractical due to the small and often environmentally variable contributions that each individual quantitative trait locus (QTL) has on total isoflavones. In this study, we developed a Magellan × PI 437654 F7-RIL population to construct a highly saturated non-redundant linkage map that encompassed 451 SNP and SSR molecular markers and used it to locate genomic regions that govern accumulation of isoflavones in the seeds of soybean. Five QTLs were found that contribute to the concentration of isoflavones, having single or multiple additive effects on isoflavone component traits. We also validated a major locus which alone accounted for up to 10% of the phenotypic variance for glycitein, and 35-37% for genistein, daidzein and the sum of all three soybean isoflavones. This QTL was consistently associated with increased concentration of isoflavones across different locations, years and crosses. It was the most important QTL in terms of net increased amounts of all isoflavone forms. Our results suggest that this locus would be an excellent candidate to target for MAS. Also, several minor QTLs were identified that interacted in an additive-by-additive epistatic manner, to increase isoflavone concentration.