Japanese Brown cattle are the second most popular Wagyu breed, and the Kumamoto sub-breed shows better daily gain and carcass weight. One of the breeding objectives for this sub-breed is to reduce genetic defects. Chondrodysplastic dwarfism and factor VIII deficiency have been identified as genetic diseases in the Kumamoto sub-breed. Previously, we detected individuals in the Kumamoto sub-breed with causative alleles of genetic diseases identified in Japanese Black cattle. In the current study, 11 mutations responsible for genetic diseases in the Wagyu breeds were analyzed to evaluate the risk of genetic diseases in the Kumamoto sub-breed. Genotyping revealed the causative mutations of chondrodysplastic dwarfism, factor XI deficiency, and factor XIII deficiency and suggested the appearance of affected animals in this sub-breed. DNA testing for these diseases is needed to prevent economic loses in beef production using the Kumamoto sub-breed.