South American horses constitute a direct remnant of the Iberian horses brought to the New World by the Spanish conquerors. The source of the original horses was Spain, and it is generally assumed that the animals belonged to the Andalusian, Spanish Celtic, Barb or Arabian breeds. In order to establish the relationship between Argentinean and Spanish horses, a portion of the mitochondrial D-loop of 104 animals belonging to nine South American and Spanish breeds was analysed using SSCP and DNA sequencing. The variability found both within and between breeds was very high. There were 61 polymorphic positions, representing 16% of the total sequence obtained. The mean divergence between a pair of sequences was 2.8%. Argentinean Creole horses shared two haplotypes with the Peruvian Paso from Argentina, and the commonest haplotype of the Creole horses is identical to one of the Andalusian horses. Even when there was substantial subdivision between breeds with highly significant Wright's Fixation Index (FST), the parsimony and distance-based phylogenetic analyses failed to show monophyletic groups and there was no clear relationship in the trees between the South American and any of the other horses analysed. Although this result could be interpreted as mixed ancestry of the South American breeds with respect to the Spanish breeds, it is probably indicating the retention of very ancient maternal lineages in the breeds analysed.