Simple SummaryIn modern times, horse breeds, mostly in horse racing, are the Thoroughbred varieties obtained by breeding three Godolphin Arabians with British mares in England. Especially in Jeju Island, Korea, Jeju horses have been introduced from Mongolia since the 13th century. They have contributed a lot to the agricultural community, but their population has been rapidly decreasing due to rapid agricultural industrialization. Therefore, we sympathize with Jeju horse-specific genetic variation and compare and analyze evolutionary correlations by utilizing Whole Genome Sequencing analysis to evaluate the genetic diversity of Jeju horses and preserve genetic information. We explored Jeju horse-specific genetic differences through a comparative analysis of large-capacity genomic data between the public database and a Thoroughbred variety. In order to adapt to the barren external environment, it is predicted that Jeju horses have experienced strong positive selection in the direction of accumulating many genetic variations, enough to cause functional differences in the eqCD1a6 gene to have an efficient immune function. In addition, we further validate the Jeju horse-specific single nucleotide polymorphisms in the aqCD1a6 gene by employing the digital PCR method, a diagnostic technique for genetic variations.The Jeju horse is a native Korean species that has been breeding on Jeju Island since the 13th century. Their shape has a distinct appearance from the representative species, Thoroughbred. Here, we performed a comparison of the Jeju horse and Thoroughbred horse for the identification of genome-wide structure variation by using the next-generation sequencing (NGS) technique. We generated an average of 95.59 Gb of the DNA sequence, resulting in an average of 33.74 X sequence coverage from five Jeju horses. In addition, reads obtained from WGRS data almost covered the horse reference genome (mapped reads 98.4%). Based on our results, we identified 1,244,064 single nucleotide polymorphisms (SNPs), 113,498 genomic insertions, and 114,751 deletions through bioinformatics analysis. Interestingly, the results of the WGRS comparison indicated that the eqCD1a6 gene contains signatures of positive natural selection in Jeju horses. The eqCD1a6 gene is known to be involved in immunity. The eqCD1a6 gene of Jeju horses commonly contained 296 variants (275 SNPs and 21 INDELs) that were compared with its counterpart of two Thoroughbred horses. In addition, we used LOAA, digital PCR, to confirm the possibility of developing a molecular marker for species identification using variant sites. As a result, it was possible to confirm the result of the molecular marker with high accuracy. Nevertheless, eqCD1a6 was shown to be functionally intact. Taken together, we have found significant genomic variation in these two different horse species.