The total genomic DNA of the male and female cycads (Cycas tanqingii D.Y. Wang) was extracted from the leaflets of half a year old using an optimized CTAB protocol. The RAPD-PCR amplification conditions were optimized by monofactor and multifactor orthogonal experiments. A total of 160 primers were used for RAPD-PCR amplification and more than 1 450 fragments were obtained from the male or the female plants. A fragment of about 500 bp only linked to the female was generated steadily by the S0465 (CCCCGGTAAC) primer, which can be used in sex determination of C. tanqingii.