Soybean seed isoflavones namely genistein, daidzein, and glycitein have numerous human health benefits such as reducing the risk of breast and prostate cancer, the symptoms of menaupose in woemn and cardiovascular diseases. Understanding the genetic control of seed isofalvones contents is of great importance to develop cultivars that produce high amounts of these isoflavones. The objective of this study was to map quantitative trait loci (QTL) for seed isoflavones contents in the ‘PI 438489B’ by ‘Hamilton’ recombinant inbred line (RIL) population of soybean. A total of six QTL for seed isoflavones content were identified 5 different linkage groups (LGs) of the soybean geneome. The QTL LOD scores ranged from 2.6 to 5.3. Two QTL were identified for daidzein: qDAID001 and qDAID002 on chromosome 2 (LG M) and chromosome 17a (LG D2), respectively. Similarly, two QTL were identified for glycitein: qGLY001 and qGLY002 on chromosome 2 (LG D1b) and chromosome 8 (LG A2), respectively. Three QTL were identified for genistein: qGEN001 on chromosome 8 (LG A2), and both qGEN002 and qGEN003 on chromosome 12 (LG H). One QTL (qGLY002/qGEN001) controls both glycitein and genistein contents. The QTL identified here can be introduced in breeding programs aimed at producing cultivars with high seed isoflavones contents.