Chalcone synthase (CHS), the key enzyme in the flavonoid biosynthesis pathway, is encoded by a multigene family, CHS1-CHS8 and dCHS1 in soybean. A tandem repeat of CHS1, CHS3 and CHS4, and dCHS1 that is believed to be located in the vicinity comprises the I locus that suppresses coloration of the seed coat. This study was conducted to determine the location of all CHS members by using PCR-based DNA markers. Primers were constructed based on varietal differences in either the nucleotide sequence of the 5'-upstream region or the first intron of two cultivars, Misuzudaizu, with a yellow seed coat (II), and Moshidou Gong 503, with a brown seed coat (ii). One hundred and fifty recombinant inbred lines that originated from a cross between these two cultivars were used for linkage mapping together with 360 markers. Linkage mapping confirmed that CHS1, CHS3, CHS4, dCHS1, and the I locus are located at the same position in molecular linkage group (MLG) A2. CHS5 was mapped at a distance of 0.3 cM from the gene cluster. CHS2 and CHS6 were located in the middle region of MLGs A1 and K, respectively, while CHS7 and CHS8 were found at the distal end of MLGs D1a and B1, respectively. Phylogenetic analysis indicated that CHS1, CHS3, CHS4, and CHS5 are closely related, suggesting that gene duplication may have occurred repeatedly to form the I locus. In addition, CHS7 and CHS8 located at the distal end and CHS2, CHS6, and CHS members around the I locus located around the middle of the MLG are also related. Ancient tetraploidization and repeated duplication may be responsible for the evolution of the complex genetic loci of the CHS multigene family in soybean.