As the largest grouper species, the giant grouper (Epinephelus lanceolatus) exhibits rapid growth and significant competitive advantages, playing a crucial role in the development of the grouper industry. To understand the impact of artificial breeding and selection activities on the genetic diversity of giant grouper, this study employed microsatellite molecular marker technology to investigate the genetic variation in five representative breeding populations collected from Guangdong, Hainan, and Fujian provinces in China. Genetic diversity analysis within the populations revealed an average number of alleles (Na) of 7.326 (range: 6.375-8.380), an average observed heterozygosity (Ho) of 0.711 (range: 0.625-0.775), an average expected heterozygosity (He) of 0.705 (range: 0.684-0.734), and an average polymorphic information content (PIC) of 0.659 (range: 0.633-0.693). Notably, the breeding population from Xiang"an District, Xiamen, Fujian exhibited the highest genetic diversity. Analysis of molecular variance (AMOVA) revealed that 5.36% of the genetic variation originated from among populations, while 95.45% originated from within individuals. The genetic differentiation index (Fst) and genetic distance results indicated that the GC and CP populations clustered together, followed by the AT population, the XA population, and the HL population forming separate branches. The phylogenetic tree analysis showed overlapping of breeding populations without distinct geographical patterns. In conclusion, the breeding populations of giant grouper in these three provinces exhibited high genetic diversity without evident signs of domestication. Overall, this study demonstrated that the breeding populations of giant grouper still maintain a high level of genetic diversity, with a low likelihood of decline due to inbreeding effects. However, the frequent occurrence of diseases and low survival rates in the breeding process may be attributed to imperfect artificial breeding techniques and inadequate aquaculture management. This research provides a theoretical basis for the genetic evaluation and artificial selection of giant grouper.