Several studies on the correlation between temperament and genetic diversity are conducted in animals as well as human. Horse temperament is especially important because it is important factor for horse riding and racing. In this study, we performed targeted exome sequencing to find single nucleotide polymorphisms (SNPs) served as genetic markers that can evaluate the aggressive and docile levels of horses. We selected 71 candidate genes related to animal and human temperament through previous researches and verified it on the human reference genome (hg38) and horse reference genome (equCab2). We found that 16 orthologous genes were present in horse reference genome and 17 homologous genes found in horses based on the human reference genome. Finally, we designed probes to find the genetic variation in selected 33 genes. The sequencing libraries were constructed using the designed probe and DNA samples extracted from the blood of 8 aggressive and 8 docile horses. The constructed libraries were sequenced using the Illumina Hiseq2500 platform. SNPs data obtained from targeted exome sequencing will be used for genome wide association study (GWAS) and Sanger sequencing validation. This study will help to assess the horse temperament and to select superior horses for riding or racing.