Soybean (Glycine max (L) Merr.) isoflavones have attracted considerable attention for their diverse effects on human health. To determine the genetic factors that contribute to the high isoflavone contents of the soybean varieties 'Peking' and 'Tamahomare', we conducted QTL analyses for the total content of daidzein derivatives (DAC), genistein derivatives (GEC) and glycitein derivatives (GLC), and for the total content of isoflavones (TIC) using recombinant inbred lines (RILs) derived from the cross between 'Peking' and 'Tamahomare'. Ninety six RILs were planted in Kyoto, Osaka and Nagano in 2003 and in Osaka and Nagano in 2004. Transgressive segregation for TIC was detected in all the environments tested. Composite interval mapping for TIC revealed four QTLs: qIso1, qIso2, qIso3 and qIso4, located on LG-A1 (Chr.5), LG-A2 (Chr.8), LG-C1 (Chr.4) and LG-D2 (Chr.17), respectively. The high-isoflavone alleles were derived from Peking at qIso1 and qIso4 and from Tamahomare at qIso2 and qIso3. Several other groups have already reported the former two QTLs, but qIso2 and qIso3 are new discoveries. Our results indicate that the large variation in TIC measurements observed in the RILs could have resulted from the combined effects of alleles at the four QTLs derived from distantly related varieties.