In this study we optimized the methods for preparing the chromosomes of fish in different sizes and analyzed the karyotypes of the chromosomes of hybrid F1 groupers (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus ♂). Juveniles of hybrid F1 groupers were pre-treated with small fish swimming method premised on the results of pre-experiments. Then we collected the fins of hybrid F1 groupers to extract the chromosomes using different drop processing methods. The morphology and karyotypes of the chromosomes were observed and analyzed under the microscopes. We obtained clear images of the chromosomes with selected measures and their mitotic indices were different. The hot air drying method generated higher-quality chromosomes than the cold air drying method. Moreover, the mitotic index after hot air drying treatment was 2.58%, which was significantly higher than the cold air drying method (0.83%). It was found that there were 44 telocentric chromosomes and 4 subtelocentric chromosomes in the diploid, and that the karyotypic formula was 2n=48, 4st+44t, and NF=52. The chromosome number and karyotype of the hybrid F1 groupers were similar to their fraternal parent, while the karyotype was great different from their maternal parent. These results demonstrated that the chromosome number of hybrid F1 groupers was the same as their parents’, and the karyotypes showed a typical pattern of the higher group of fish evolution taxonomy. Furthermore, we also compared the karyotypes of the related 25 species of groupers. Our study will provide scientific guidance for the germplasm identification, genetic resources protection and breeding of the groupers