The phylogenesis of thirty-three tobacco germplasms belonging to different brown spot resistance analyzed by six SSR markers which linked to the tobacco brown spot resistant genes and twenty-one random SSR markers were reported in this study.The results of cluster analysis based on the six tobacco brown spot resistant linked SSR markers flanking the three tobacco brown spot resistant QTLs showed that thirty-three tobacco varieties could be clustered into four groups at a relative low level of GS (genetic similarity coefficient) 0.44.The tobacco varieties which had the resistant resource to tobacco brown spot from the Jingyehuang (China) and Beinhart-1000 (USA) were clustered into group Ⅰ;the tobaccos which belong to CV series and are resistant to tobacco brown spot were clustered into group Ⅱ;N.suaveolens belonging to wild tobacco was in an unique group Ⅳ;while the left which belong to susceptible varieties such as Changbohuang,G140,and NC82 were clustered into group Ⅲ.The results of cluster analysis showed that the SSR markers linked to the tobacco brown spot resistant genes (flanking the resistant QTLs) could cluster the species into resistant and susceptive while the random SSR makers could not.