Genetic diversity is the cornerstone of species survival and evolution. For most aquatic animals, keeping the high genetic diversity is the guarantee of population life ability. Groupers are important economic fishes for mariculture due to delicious taste and rich nutrition. With the rapid development of grouper breeding technology, the artificial culture of grouper has become the second marine fish industry of China in 2019. The hybrid grouper [Epinephelus fuscoguttatus(♀)× E. lanceolatus(♂)] is the maximum yield aquaculture grouper with heterosis of growth and disease resistance. In recent years, more and more culture groups showed growth phenotypes variation and frequent diseases. The decline of genetic diversity and inbreeding depression are considered to be the common reasons for the decline of aquaculture bio-economic traits. In order to explore whether the decrease of population genetic diversity leads to the decrease of breeding traits of hybrid grouper, in this study, 27 EST-SSRs markers were used to analyze the genetic diversity and population genetic structure of 6 groups of the hybrid grouper in the main seed production area. The results showed that, the average number of alleles (N_a) ranged from 6.666 7 to 8.5556, the average number of effective alleles (N_e) from 3.511 6 to 3.959 0, and the average observed heterozygosity (H_o) ranged from 0.764 6 to 0.7943, the average expected heterozygosity (H_e) from 0.7091 to 0.750 4, average polymorphism information content (PIC) ranged from 0.662 2 to 0.706 4. The results of molecular analysis of variance (AMOVA) showed that the variances among the groups, individuals within the group, and all individuals of the cultured groups of hybrid grouper were all at a significant level (P<0.01), and principal coordinate analysis (PCoA) showed the difference mainly came from the individual. F_st between groups, genetic distance and cluster analysis showed that the HB and XC groups first clustered together, then clustered together with WT and HW groups, GC and QT groups first clustered together, FP groups were independent. Genetic structure analysis showed that all samples were divided into 5 theoretical groups (K=5). HB group was composed of FP, WT, XC and GC groups. The results of this study showed that the hybrid grouper population still had high genetic diversity, with clear grouping. The possibility of decline of hybrid varieties affected by parental inbreeding was not high. The variation of pathogenic organisms and irregular breeding management may be the causes of frequent diseases. Therefore, it is necessary to strengthen the management of non-toxic parent screening, aquaculture water and diseased fish, and cut off the vertical and horizontal transmission of viruses to achieve effective disease prevention and control. This study provides a theoretical basis for further genetic breeding and disease control of grouper.