Abstract: Microsatellite-anchored fragment length polymorphism(MFLP) is a new technique that detected the genetic variations of restricted fragments and microsatellites for DNA fingerprinting. On basis of MFLP principle, a fluorescent MFLP technique for tobacco was established in which the SSR-anchor primers were tailed by the universal M13 primer labeled with IRD fluorescent dye. 15 of 144 primer combinations which are suitable for tobacco were chosen for selective amplification, and these primer pairs were used for genetic diversity analysis of 32 tobacco varieties collected from different countries. The results showed that polymorphisms for the primer combinations ranged from 2 to 13, with an average of 4.3 among 32 tobacco varieties, the genetic similarity for these varieties ranged from 0.4 to 0.83. Cluster analysis and principal coordinate analysis (PCA) based on MFLPs provided the proper estimate of genetic relationships among varieties. These results indicate that the fluorescent MFLP technique can effectively detect DNA polymorphisms among tobacco germplasm, and it is suitable for the analysis of genetic characteristics in tobacco.