In order to understand the genetic ancestry and mitochondrial DNA (mtDNA) diversity of current Colombian horse breeds we sequenced a 364-bp fragment of the mitocondrial DNA D-loop in 116 animals belonging to five Spanish horse breeds and the Colombian Paso Fino and Colombian Creole cattle horse breeds. Among Colombian horse breeds, haplogroup D had the highest frequency (53%), followed by haplogroups A (19%), C (8%) and F (6%). The higher frequency of haplogroup D in Colombian horse breeds supports the theory of an ancestral Iberian origin for these breeds. These results also indicate that different selective pressures among the Colombian breeds could explain the relatively higher genetic diversity found in the Colombian Creole cattle horse when compared with the Colombian Paso Fino.