Key factors affecting the digestion-ligation,pre-amplification and selective amplification in amplified fragment length polymorphism(AFLP) analysis in sesame (Sesamum indicum L.) were optimized.Results showed that the best protocol was 200 ng/μL template DNA，digestion-ligation at 37℃ for 6 h,1.0 mmol/L Mg2+,0.3 mmol/L dNTP,0.5 U Taq DNA polymerase in the pre-amplification system and pre-amplification products diluted by 10 times,1.0 mmol/L Mg2+, 0.2 mmol/L dNTP,and 1.0 U Taq DNA polymerase,0.6 mmol/L primers in the selective amplification.