Yellow grouper Epinephelus awoara is a economically important marine fish mainly distributed in Pacific-northwest , Pacific-western central and South China, but the number of its population extremely decreased due to overfishing of grouper adults and juveniles, habitat damage and loss from bottom trawling, pollution, high demand for food and so on these years. E. awoara was listed in the 2005 IUCN Red List as a specie with DD(data deficient). Since molecular markers are very useful tools to estimate germplasm resources, variety identification, genetic diversity analysis and preparation of genetic linkage map, the microsatellites of E. awoara were isolated and thus its micosatellite markers and genetic diversity were analysed. Firstly a library of partial small size fractionated genomic DNA was constructed with the E. awoara from South China Sea. 96 microsatellites in 28 recombinant positive clones were obtained through PCR screening the library with M13 universal primers and simple tandem repeats primer (CA)15. Among these microsatellites, there were 39 perfect ones (40.6%), 30 imperfect ones (31.3%), 7 compound perfect ones (7.3%) and 20 compound imperfect ones (20.8%). The results indicated that microsatellite sequences characterized by (CA/GT)n were abundant in genomic DNA of E. awoara. 26 of 28 pairs of primers which were designed to analyze genomic DNA of E. awoara according to unique microsatellite flanking sequences with the software Primer 5.0 could be amplified expected bands, and of which 13 microsatellite markers were assessed genetic diversity for 19 individuals. As a consequence, the data showed that the average observed heterozygosity (Ho), average expected heterozygosity (He), average polymorphism information content (PIC) and average Hardy-Weinberg departure value (D) of E. awoara was 0.5982, 0.5080, 0.4722 and 0.1503 respectively. The findings indicated that although the genetic diversity of E. awoara in South China Sea was rich, but it had been influenced by human behaviors to a certain extent.