Coral trout, Plectropomus leopardus, is one of the high-priced groupers. However, wild populations of P. leopardus are facing population decline due to overharvesting and habitat destruction. In order to restore the wild populations, we established a breeding program to artificially propagate P. leopardus, and subsequently examined the genetic diversity and differentiation among the breeding stock, artificially bred stock (F1), and wild populations around the Penghu Islands of Taiwan. A total of 127 individual specimens of P. leopardus, including 60 individuals from the breeding stock, 34 F1 fish, and 33 fish from the wild populations were examined using 8 nuclear microsatellite (MST) markers. The results of the MST analyses revealed that the number of alleles (A) ranged from 3 to 20, the observed heterozygosity (Ho) ranged from 0.18 to 0.81, and the expected heterozygosity (He) ranged from 0.21 to 0.88. The allele and heterozygosity results revealed that the genetic diversity of the F1 fish was not significantly decreased in comparison to that of the breeding stock and wild populations. However, the pairwise population differentiation index (Fst) ranged from 0.021 to 0.087, and all the population pairs were significantly different (p < 0.05) from each other. Furthermore, the genetic structure analysis results revealed that distinct genetic structures existed among the breeding stock, F1, and wild populations (when k = 3 with maximum likelihood value = ‐3258.7), suggesting the effect of artificial selection on the breeding stock. Our results will be useful for the future management of the breeding stock and the conservation of coral trout in general.