Abstract: We used 11 pairs of microsatellite loci to investigate the genetic polymorphism of two breeding populations of golden pompano (Trachinotus ovatus) in Sanya, Hainan and Daya Bay, Guangdong. The average numbers of alleles (N_a) at each microsatellite locus of the populations were 5.0 and 3.6, respectively; the average numbers of valid alleles (N_e) were 2.965 and 2.244, respectively. The observed heterozygosities (H_o) were 0.292~0.814 and 0.207~0.840, respectively; the expected heterzygosities (H_e) were 0.307~0.813 and 0.189~0.761, respectively. The PIC value ranged from 0.014 to 0.719 in Sanya population with an average of 0.509, while that of the Daya Bay population ranged from 0.168 to 0.711 with an average of 0.436. In Sanya population, five loci deviated from Hardy-Weinberg equilibrium (P < 0.05), while only two loci deviated from Hardy-Weinberg equilibrium in Daya Bay population. The Nei′s genetic similarity coefficient of the two populations was 0.561 and their genetic distance was 0.578. The genetic differentiation index of the two breeding populations (F_st) was 0.152. Structure analysis indicates that the two T.ovatus populations, which had higher genetic differentiation, were divided into two groups and could be managed as two breeding populations. Good genetic progress could be achieved by hybridization between the two breeding populations.