We characterized the SNP 53 JPN System for parentage verification during horse registry. The SNP 53 JPN System was constructed using 53 highly polymorphic single nucleotide polymorphisms (SNPs), which were amplified and genotyped with 2 multiplex assays. The SNP 53 JPN System showed good resolution for 95 unrelated thoroughbreds, and the exclusion probability (PE01) for each SNP ranged from 11.5 to 23.0%, resulting in a total PE01 value of 99.996%. These results indicate that the SNP 53 JPN System is useful for parentage testing of thoroughbreds. Of the 53 SNPs, 8 SNPs could be used to exclude a pseudo parent and sib combination found using the 2006 International Society for Animal Genetics (ISAG) horse comparison test, as efficiently as the parentage testing systems using short tandem repeats (STRs). Thus, we concluded that the SNP 53 JPN System could provide sufficient and reliable information for routine parentage testing of thoroughbred.