The golden mussel (Limnoperna fortunei), a mollusc originating from Asia, has caused considerable environmental and economic damage in various Brazilian river basins and reservoirs. Genetic studies that focus on characterizing its invasive potential and the feasibility of control measures are required. The present study identified and characterized 13 new microsatellite markers (of which eight were polymorphic) in L. fortunei using enriched genetic libraries. The analysis performed on 48 individuals from three populations identified 32 alleles, ranging from one to seven alleles per locus. The size of the fragments ranged from 172 to 381 bp. The polymorphic information content values ranged from 0.114 to 0.768, with observed and expected heterozygosity values ranging from 0.043–0.938 to 0.123–0.805, respectively. Six loci showed significant deviation from the Hardy–Weinberg equilibrium, possibly influenced by the heterozygous deficit. The microsatellite primers developed will be useful in studies of the genetic diversity and population structure of L. fortunei and will also contribute to the adoption of control measures for this invasive species in Brazilian and other South American basins.