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The use of DNA technology for parentage testing is increasing every day. Most laboratories have improved their exclusion probabilities (PE) by the addition of DNA microsatellite loci to standard blood-typing results. The efficacy of each locus depends on the number of alleles detected and their frequencies in the breeds tested. Here we analyzed the usefulness of six microsatellites for routine parentage testing in three Portuguese autochthonous horse breeds: Lusitano, Sorraia and Garrano. The DNA loci analyzed - ASB2, HMS3, HMS7, HTG4, HTG10, and VHL20 - were chosen based upon the polymorphism detected in other breeds with Iberian horse influence. The estimated probability of exclusion of wrongly named parents (PE) was high, with values ranging from 88.5% to 99.6%.