Bovine claudin-16/paracellin-1 (CL-16/PCLN-1) deficiency is an autosomal recessive disorder caused by a 37-kb deletion mutation containing the first four exons of the CL-16 gene, which leads to the absence of the CL-16 transcript (type-1 mutation). A PCR-based DNA test for the CL-16 mutation (type-1) was used to screen a herd of Wagyu cattle. A recent report suggested that affected cattle can be bred by dams diagnosed as normal, suggesting the presence of a new mutation in the CL-16 locus. We identified the new mutation as a 56-kb deletion containing exon-1 to -4 and 21-bp of exon-5 of CL-16, and refer to this as a type-2 mutation. A DNA test for specific for this mutation was then established.