Epinephelus bleekeri is important in commercial fisheries of tropical and subtropical seas. It has been broadly cultured in many Asian countries, such as China, Japan, Vietnam and Malaysia.Despite many studies on its culture techniques, the detailed cytogenetic data of this species is still lacking. To add E. bleekeri cytogenetic data and lay the foundation for grouper chromosome evolution and breeding studies, the karyotype of E. bleekeri was studied in this paper using multiple methods, including Giemsa staining, Ag-NORs and C-banding. The following results were obtained: E. bleekeri had a diploid chromosome number of 48 and its karyotype formula was 48t, NF=48, and sex chromosome was not observed; the constitutive heterochromatin was observed at the centromeric region on most chromosomes; but only a single pair of NORs was detected at the paracentromeric region of chromosome pair No.24. Based upon above results, it is concluded that E. bleekeri was relatively primitive among genus Epinephelus.