The present study demonstrates a new approach that enables effective horse parentage testing using 22 ISAG microsatellite markers involving 6 heads of Thoroughbred horse(TB) and non-TB. In the comparison allele distribution between these horses, the alleles found in the TB were numerously detected in the non-TB. As results, we confirmed that these ISAG microsatellite markers might apply the pedigree registration of Korean native horse(Jeju horse).