© 2016 The Author(s).The colonial tunicate Didemnum perlucidum has been identified from numerous tropical and temperate locations worldwide, but its native and introduced ranges remain largely unknown. In Australia, D. perlucidum is listed as a target species under the introduced marine pest national monitoring network. In order to investigate the introduced status and potential routes of introduction and dispersal of D. perlucidum, we developed 17 new polymorphic microsatellite markers using 454 shotgun sequencing. Two to six alleles per locus were detected. No evidence of linkage disequilibrium between pairs of loci was identified and 12 of the 17 loci were in Hardy-Weinberg equilibrium.