AFLP(amplified fragment length polymorphism)was applied to identify markers associated with Cannabis sativa L.sex type. Sixty four pairs of primers of the 8 EcoRⅠ-NNN and 8 MseⅠ-NNN combination were used to amplify the mixed male and female DNA pools of ten varieties. The primer E-ACT/M-CTA amplified an apparent fragment that was only existed in male pool and in all male individuals of the ten varieties. The male-specific fragment was 348 bp after recovering, cloning and sequencing. None of its homologous sequence was found in GenBank. Therefore, the male-specific fragment can be used as linkage marker in sex identification of Cannabis sativa .