In soybean [Glycine max (L.) Merr.], the dominant allele of the T locus for tawny pubescence is associated with chilling tolerance and is useful for breeding for chilling tolerance. Yellow hilum cultivars with the dominant I allele are preferred in Japan because of a better external appearance. However, the II TT allelic combination darkens the entire seed coat and degrades the external appearance. This study was conducted to investigate the genetic basis of seed coat discoloration using DNA markers. F-2 populations derived from crosses between soybean breeding lines 0518BW-8 with slight discoloration and 0734BW-1 with severe discoloration were grown at Memuro in 2003 and Kunneppu in 2007. Seed coat discoloration was measured by the L* value with a two-dimensional colorimeter. A total of 104 simple sequence repeat markers were classified into 20 linkage groups (LGs) spanning 1025 cM in 2003 and 19 LGs spanning 789 cM in 2007. A quantitative trait locus (QTL), discol1, was identified by composite interval mapping in LG A2 (chromosome 8) across years and locations. It had an LOD score of 5.58 explaining 22.0% of phenotypic variance in 2003 and a LOD score of 5.62 explaining 20.2% of variance in 2007. The allele from 0518BW-8 increased the L* value at the QTL. Position of discol1 was close to the / locus corresponding to the chalcone synthase (CHS) multigene family cluster. No sequence differences were observed between the two lines in nucleotide sequences of CHS1 and CHS3 genes and the intergenic region between CHS1 and CHS3.