To investigate the mechanism of sex determination of crabs, amplified fragment-length polymorphism DNA analysis was applied to identify sex-specific markers for Portunus trituberculatus. Eleven pairs of primers were used to amplify DNA isolated from male and female crabs. A total of 481 and 499 fragments were amplified for females and males, respectively, and the ratios of polymorphic fragments were 20.59-65.52% and 27.27-62.22%, respectively. Distribution difference values between female and male groups for 10 polymorphisms were greater than 40%; Cluster analysis revealed that Nei's genetic distances for 57 polymorphisms clustered according to sex. The results indicate that a difference at the DNA level exists between female and male crabs and provide data for further study on sex determination.