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Jeju horse (Natural Monument number 347) is a breed of horse that has experienced long-term isolation and domestication in Jeju Island, South Korea. We evaluated genetic features of this breed, including SNP, by whole-genome resequencing using an Illumina HiSeq 2000. A total of 5,986,852 SNP were identified in 4 Jeju horses and were divided into homozygous and heterozygous SNP (2,357,099 and 3,629,753 SNP, respectively). It revealed that 63.8% of these SNP resided in intergenic regions. Immune response genes with nonsynonymous SNP were overrepresented in Jeju horses as evidenced by Gene Ontology clustering. Among these genes, Toll-like receptors (TLR) are highly enriched. Comparing TLR genes between Jeju horses and the Przewalski's horse, and genes showed "possibly damaging" mutations in several regions by analysis with PolyPhen-2. These results provide a framework for further genetic studies in Jeju horse by domestication. Furthermore, research on functions of SNP-associated genes would aid in understanding the molecular genetic variation of horse breeds.