Soybean seeds contain large amounts of isoflavones or phytoestrogens such as genistein, daidzein, and glycitein that display biological effects when ingested by humans and animals. In seeds, the total amount, and amount of each type, of isoflavone varies by 5 fold between cultivars and locations. Isoflavone content and quality are one key to the biological effects of soy foods, dietary supplements, and nutraceuticals. Previously we had identified 6 loci (QTL) controlling isoflavone content using 150 DNA markers. This study aimed to identify and delimit loci underlying heritable variation in isoflavone content with additional DNA markers. We used a recombinant inbred line (RIL) population (n=100) derived from the cross of “Essex” by “Forrest,” two cultivars that contrast for isoflavone content. Seed isoflavone content of each RIL was determined by HPLC and compared against 240 polymorphic microsatellite markers by one-way analysis of variance. Two QTL that underlie seed isoflavone content were newly discovered. The additional markers confirmed and refined the positions of the six QTL already reported. The first new region anchored by the marker BARC-Satt063 was significantly associated with genistein (P=0.009, R2=29.5%) and daidzein (P=0.007, R2=17.0%). The region is located on linkage group B2 and derived the beneficial allele from Essex. The second new region defined by the marker BARC-Satt129 was significantly associated with total glycitein (P=0.0005, R2=32.0%). The region is located on linkage group D1a+Q and also derived the beneficial allele from Essex. Jointly the eight loci can explain the heritable variation in isoflavone content. The loci may be used to stabilize seed isoflavone content by selection and to isolate the underlying genes.