Estuary perch (Percalates colonorum Gunther) is an estuary dependent fish native to south-eastern Australia that is in decline. There is an increasing emphasis on stocking the species. Understanding the genetic structure across its range is important for guiding optimal stocking strategies. A prior study found some evidence of population genetic structure; however, few genetic markers were used in that assessment. Here, we develop 21 novel polymorphic microsatellite markers to reassess population genetics. Analyses indicate three broad genetic clusters, with populations on mainland Australia exhibiting an isolation by distance pattern. The only known population from Tasmania is genetically and geographically isolated from mainland populations and has very low levels of genetic diversity. We provide recommendations for sourcing broodstock from mainland populations, including describing three broad areas for procuring and releasing broodstock and offspring. The markers and results reported here will prove invaluable for guiding and monitoring the outcomes of stocking and conservation activities.