The Chinese softshell turtle exhibits ZZ/ZW sex determination. To identify the sex of embryos, juvenile and adult individuals, we designed two pairs of polymerase chain reaction primers, SB1-196, which amplifies a fragment of 196 bp in the female and the other, CK1-482, which amplifies the 482-bp fragment in both the sexes. It is validated in 24 adult turtles of known sex, sampled from three different locations. This one-step sexing technique is rapid and easy to perform and is reported for the first time.