Pterygoplichlhys pardalis is an important freshwater ornamental fish in the Amazon Basin. Studies involving populations of P. pardalis are of great importance for the conservation and management of this species. We developed nine microsatellite loci and applied them to investigate the genetic variation of 20 wild individuals from floodplain lakes of the Solimões river. The number of alleles per locus ranged from 2 to 12, with an average of 6.6. The observed and expected heterozygosity values ranged from 0.400 to 0.923 (average 0.706) and from 0.358 to 0.895 (average 0.692), respectively. The value of f ranged from −0.532 to 0.467 (average 0.032). One locus (Pp07) significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P: (5 %)