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The 55-million-year history of equine phylogeny has been well documented from the skeletal record, however not the soft tissue structures that are now vestigial in modern horse. A recent study reported 2 ligamentous structures resembling functional 3rd and 4th interosseous muscles were evident in Dutch Konik horses. The current study investigates this finding and compares it to members of the genus Equus to identify either a breed anomaly or functional primitive trait. Distal limbs (n=571) were dissected from 4 species of Equus; E. caballus, E. asinus, E. przewalskii and E. burchelli beohmi. Breed representatives of E.caballus (n=18) included the primitive Dutch Konik. The 2nd and or 4th interosseous muscle was evident in all 4 species, but only 2 breeds of E.caballus expressed this trait - the Dutch Konik and Bosnian Mountain Horse. These 2 breeds were the only close descendants of the extinct Tarpan (Equus ferus ferus) represented in this study. In conclusion, the 2nd and 4th interosseous muscles originated from the distal nodule of respective splint bones and inserted into the corresponding branches of the 3rd interosseous muscle proximal to the sesamoids. Suggesting a functional role in medial and lateral joint stability and a primitive trait in modern equids.