This study details 13 novel polymorphic microsatellite loci in the armoured catfish Hypostomus ancistroides, and assesses their utility for population genetic studies. The analysis of 30 individuals revealed a total of 99 different alleles (ranging from two to 15 alleles per locus), with an average of 7·62 alleles per locus, with observed and expected heterozygosities ranging from 0·103 to 0·931 and from 0·102 to 0·906, respectively. One of the 13 loci showed significant deviations from Hardy-Weinberg equilibrium, probably due to the presence of null alleles, inferred from the excess of homozygotes.