The objective of the present study was to investigate the genetic diversity and population genetic structure of the commercially important longfin grouper, Epinephelus quoyanus, which is widely distributed in the western Pacific Ocean. A total of 74 individuals were sampled from the waters of northern Taiwan (NT), Penghu (PH) and southern Great Barrier Reef (GBR), Australia (AUS), from 2011-2013. A total of 14 microsatellite loci were used to determine the population genetic diversity and structure of these 3 populations. The results from the selected microsatellites showed that the genes for the 3 populations were polymorphic. NT and PH had 9 and 8 microsatellite loci, respectively, and the heterozygosity observed was higher than the heterozygosity expected. FIS values ranged from -0.165 to 0.403, with no significant differences among the populations. The pairwise population differentiation index (Fst) values ranged from 0.0018 to 0.0715, with no significant differences between the groups, indicating that there was low population differentiation among the 3 populations. The result of Bayesian-clustering assignment test revealed that the sampled groupers can be regarded as two populations (when k =2, it has the highest maximum likelihood value) based on allele frequencies of all sampled individual. There were several common alleles among the 3 populations, supporting the conclusion that there was low population differentiation among the 3 populations and that the NT and PH populations should be viewed as a single population for the purposes of fishery resource assessment and management.