Abstract: The methods of CTAB,improved CTAB and SDS were used for genomic DNA isolation of climax leaves and young leaves of Wintersweet cultivars.The results indicated that the methods of CTAB was useful in Wintersweet cultivars genomic DNA isolation,and the leaf age doesn’t affect genomic DNA isolation of Wintersweet cultivars.By the amplified fragment length polymorphisms(AFLP) technique,using MseI-EcoR I enzyme-cut combination,ten pairs of primers(i.e.M23E46,M24E46, M25E46,M23E47,M24E47,M41E47,M41E94,M64E94,M64E66 and M24E75) with high polymorphism and powerful distinctiveness were selected from 168 pairs of primer,and the best pre-expanding and selective expanding system were determined.Our results tentatively established the basis for the research of the cultivar classification and gentic diversity analysis.