We isolated 25 new polymorphic microsatellite markers from the eastern mosquitofish, Gambusia holbrooki. Initially, 454 shotgun sequencing was used to identify 1187 loci for which primers could be designed. Of these 1187, we trialled 48 in the target species, 40 of which amplified a product of expected size. Subsequently, those 40 loci were screened for variation in 48 individuals from a single population in Canberra, Australia. Twenty loci were in Hardy–Weinberg equilibrium and polymorphic, with observed heterozygosity ranging from 0.04 to 0.72 (mean: 0.45 ± 0.18) and the number of alleles per locus ranged from 2 to 5 (mean: 3.20 ± 1.05). These loci will be useful in understanding genetic variation, paternity analysis and in managing this species across both its native and invasive range.