Abstract: In this study, MISA software was used to screen and analyze the microsatellites in the four genomes of puffer fish. As a result, in the genome of Takifugu rubripes（391.49 Mb）, T. flavidus（366.29 Mb）, T. bimaculatus（371.68 Mb） and Tetraodon nigroviridis（342.40 Mb）, 142 885, 135 009, 147 549 and 179 703 perfect microsatellites were screened, respectively. The relative frequency were 365/Mb, 369/Mb, 397/Mb and 525/Mb, respectively. The total lengths of the microsatellites were 2 876 322 bp, 2 689 710 bp, 3 140 445 bp, and 3 615 069 bp, respectively,accounting for 0.73%, 0.73%, 0.84% and 1.06% of the total length of the genome sequence. Among the complete microsatellites with 1～6 different base repeat types, the orders of 6 base types from the four species of puffer fish were the same. Each had the largest number of mononucleotide repeats, followed by dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats, and hexanucleotide repeats. Among them, AC,A, C, AG, AGG, AT, AAT and AAC were highest frequency core repeat categories, which were shared by the four species of puffer fish. The distribution characteristics of the microsatellites from the three species of Takifugu were very similar. The genetic distances of T. rubripes and T. bimaculatus might be closer. Tetraodon nigroviridis was similar to the other three species of genus Takifugu, except for some microsatellite features. There were big gaps in the total number of microsatellites, the relative frequency and density of microsatellites, the number and type of partial microsatellites types in the four species, which might be related to the geographical distribution of two genus and the base composition of sliding replication. It was speculated that the genomes of Takifugu and Tetraodon might have unique evolutionary mechanisms. This study laid a foundation for the analysis of four species of puffer fish, the design of the four species of puffer fish microsatellite primers, the genetic distance of different genus species, and the relationship between phylogenetic relationships.