Previous studies revealed that the recessive allele of the W2 locus generated purple-blue color and high vacuolar pH of flower petals in soybean. The location of W2 gene was reportedly close to simple sequence repeat marker Satt318 in molecular linkage group B2. We used information from the soybean genome to clone a candidate gene for W2. An MYB transcription factor gene belonging to G20 group was found in the vicinity of Satt318. Full-length cDNAs were cloned from purple-flowered cultivar Harosoy (W2 allele) and purple-blue flowered cultivars, Nezumisaya and w2-20 (w2 allele), by reverse transcription-PCR and designated as GmMYB-G20-1. Its open reading frame was 1083 bp long that encoded 361 amino acids in Harosoy. GmMYB-G20-1 had 53.7% similarity in amino acid sequence with the PH4 gene of petunia controlling blueness and vacuolar pH of flower petals. GmMYB-G20-1 of Nezumisaya and w2-20 had 3 base substitutions compared with that of Harosoy. The first substitution generated a stop codon in the MYB domain, resulting in truncated polypeptides. Cleaved amplified polymorphic sequence (CAPS) marker was developed to detect the base substitution. The polymorphic CAPS marker co-segregated with alleles at the W2 locus in the F(2) population. These results suggest that GmMYB-G20-1 might correspond to the W2 gene.