Abstract: ISSR marker was used to find genetic relationship between 12 local tobacco varieties (strains) and 5 major tobacco varieties of Guizhou in this study. The result showed that 23 ISSR primers tested could produce distinct fingerprints among 17 varieties (strains) of tobacco. An acceptable DNA polymorphic fingerprints was constructed with 9 primers which could be used to identify these tobacco varieties (strains), all varieties (strains) were distinguished from each other and showed genetic diversity. The result from the analysis were used by NTSYS 2.0. The result showed that genetic similarity of these cultivated varieties(strains) from 0.212 7 to 0.787 2 by SimQual and these 17 varieties (strains) of tobacco could be divided into three groups by UPGMA clustering analysis.