In the present study, typeⅠmicrosatellites of Portunus trituberculatus were screened from 13985 ESTs in the NCBI database by bioinformatics approach. A total of 287 type Ⅰ microsatellites were identified, among which 173 were the type of dinucleotide repeat being the most abundant microsatellite and accounting for 60.3% of the identified microsatellite markers, and 79 were the type of trinucleotide accounting for 27.5%, while hexa-, penta- and tetra-nucleotide repeats were rare. Among the dinucleotide repeats, AC/GT was the most abundant type with percentage of 53.8%, followed by the AG/CT type with 37.0%, while AT and GC were rare. 14 microsatellites were selected for polymorphism analyses, 8 of them showed polymorphism in a wild population of Portunus trituberculatus. The mean PIC and heterozygosity values were 0.57 and 0.63, respectively, among them, 6 PIC of microsatellites greater than 0.5, showing high polymorphism. This study showed that the way of screening typeⅠmicrosatellite markers from EST data was feasible. These typeⅠmicrosatellites can be used for further study in genetic diversity, genetic mapping and quantitative trait loci (QTLs) identification in Portunus trituberculatus.