In the present study, amplified fragment length polymorphism (AFLP) was used for the isolation of sex associated markers in the genome of Chinese mitten crab, 〖WTBX〗Eriocheir sinensis〖WTBZ〗. By screening the male, female and male/female mix bulks with 192 selective AFLP primer combinations, we obtained 5 376 polymorphic fragments in total, each combination generated 28 fragments on average. A total of 88 sex difference AFLP candidates were retrieved. Subsequently, the candidates were further verified in 10 female and 10 male individuals by AFLP again. Sixty two sex difference fragments were obtained finally, and they could be classified into two groups: (1) Thirteen bands were present in four to six male crabs and absent in all female crabs; (2) Forty nine bands were present in four to six female crabs, but absent in all male crabs. No sex difference was found in the other 26 bands. The sex difference fragments were recovered, cloned and sequenced. The results showed that multiple sequences presented in some bands. Seventy seven DNA sequences were obtained, thirty one of which shared partial homology with sex chromosomal DNA sequence of birds, amphibians and mammals. The length of identical sequences ranged from 22 to 212 bp. Among them, six sequences had biological relevance with hit sequences. The SCAR (sequence characterized amplified region) primers were designed to identify sex specific SCAR marker. However, none of the SCAR derived from the presumed sex associated fragment showed sex specificity.