The variable number of tandem repeats (VNTR) polymorphism of the dopamine D4 receptor (DRD4) gene has been reported to be associated with the personality trait of novelty-seeking in humans. In the genus Equus, this region includes an 18-bp repeat unit and there are inter- and intraspecies differences in the number of repetitions. Because horses are unique among livestock species in that their temperament is considered important, we investigated the possible role of this region on equine temperament in thoroughbred horses. We simultaneously determined the sequences of this polymorphic region and administered a questionnaire survey to horse caretakers with questions about 20 different traits of their horses' temperament. Although there was no difference in the number of repeats among the 136 thoroughbred horses studied, two single nucleotide polymorphisms (SNPs), one of which might cause an amino acid change (A-G substitution), existed. By analyzing the association between these SNPs and temperament scores, a significant association was revealed between two temperament traits (Curiosity and Vigilance) and the A-G substitution. Horses without the A allele had significantly higher Curiosity and lower Vigilance scores than those with the A allele at the A-G substitution. In addition, similar associations between both temperament scores and each genotype of the A-G substitution were observed in two subgroups divided according to the time of their introduction to the farm. These results suggested that the SNP in the VNTR region of the equine DRD4 gene might be related to individual differences in equine temperament.