In the 1980s, a conservation programme involving a feral horse population, the Retuertas horses from the Guadalquivir marshes, was started in the Doñana National Park. The analysis of an extensive genetic survey of this population, which now numbers 100 animals, and 10 additional European and North African breeds using DNA polymorphisms from 22 microsatellites is presented. Highly significant fixation indexes were obtained for all pairwise comparisons between the Retuertas population and other breeds. A population neighbour-joining breed phenogram was built using different distance measures, but the Retuertas population failed to cluster with either of the two major clades of European and North African breeds, highlighting its uniqueness. In fact, the Retuertas population was positioned at the base of the trees, which were rooted using donkey samples. Furthermore, assignment tests and the individual Q-matrices obtained with the structure programme isolated the Retuertas breed from the other breeds with only four K groups. Interestingly, some local semi-feral horses, known as Marismeño, also currently living in the Guadalquivir marshes, have some microsatellite genotypes that fall well within the Retuertas cluster. This raises the possibility of incorporating horses from the Marismeño population in a future conservation programme.