Unlike other European domesticates introduced in the Americas after the European invasion, equids (Equidae) were previously in the Western Hemisphere but were extinct by the late Holocene era. The return of equids to the Americas through the introduction of the domestic horse (Equus caballus) is documented in the historical literature but is not explored fully either archaeologically or genetically. Historical documents suggest that the first domestic horses were brought from the Iberian Peninsula to the Caribbean in the late 15th century CE, but archaeological remains of these early introductions are rare. This paper presents the mitochondrial genome of a late 16th century horse from the Spanish colonial site of Puerto Real (northern Haiti). It represents the earliest complete mitogenome of a post-Columbian domestic horse in the Western Hemisphere offering a unique opportunity to clarify the phylogeographic history of this species in the Americas. Our data supports the hypothesis of an Iberian origin for this early translocated individual and clarifies its phylogenetic relationship with modern breeds in the Americas.