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The Miyako horse is one of the eight Japanese native horse breeds and is scarce in terms of population size. Japanese native horses are believed to be descended from Mongolian horses and represent a precious lineage that has preserved its traits for over a thousand years in Japan. Genetic analysis of the endangered Miyako horse is useful for preserving its original phenotypic traits. This study analysed the genetic variation in trait-related genes for coat colour (<i>ASIP, MC1R</i>), body composition (<i>MSTN, LCORL</i>), gait (<i>DMRT3</i>), and temperament (<i>HTR1A</i>) in 46 extant Miyako horses. In addition, because numerous Miyako horses have vertical stripes on their backs, we analysed the genes associated with the dorsal stripe (<i>TBX3</i>), a characteristic of primitive horses. Until now, Miyako horses were believed to comprise only bays and chestnuts, but genetic analysis revealed that two individuals were black. No mutations were detected in <i>MSTN</i> g.66619237delinsSINE, <i>LCORL</i>, or <i>DMRT3</i>, whereas polymorphisms were observed in <i>MSTN</i> g.66608679T>C and <i>HTR1A</i>. Furthermore, <i>TBX3</i> analysis suggested that the patterns observed on the backs of Miyako horses could be genetically classified as dorsal stripes. Using the information on trait-related genes obtained in this study as a foundation for breeding plans is expected to contribute significantly to the production of individuals that maintain the phenotype traditionally preserved in Miyako horses. Furthermore, genetic confirmation of the Miyako horse dorsal stripe pattern could provide significant clues regarding similar markings in other Japanese native horses.