White spotting (WS) is one of the defective appearances in Japanese Black cattle, and can be not only obstacles for improvement of the breed but also cause economic losses of farmers. However, a DNA marker linked to WS has not been found yet in Japanese Black cattle. In this study, we investigated a relationship between a MITF gene SNP (g.32386957 A&gy;T ) related to WS in foreign breeds (Fontanesi et al . 2012) and WS in Japanese Black cattle bred in Kagoshima Prefecture. Forty non-spotted and 39 spotted DNA samples were used for genotyping. Genotyping was performed by either direct sequencing or allele-specific PCR. As a result, we observed both alleles of the g.32386957 A&gy;T SNP in this population. Furthermore, composition of the SNP genotypes was clearly different between non-spotted and spotted groups (P =1.53 × 10-6), suggesting that the g.32386957 A&gy;T SNP was also strongly associated with WS in Japanese Black cattle. Variation in the MITF gene clearly explained the differences between nonspotted and spotted phenotypes but, at the same time, it was also evident that this SNP is not completely associated with the WS in Japanese Black cattle.