Abstract: For better understand the relationship between the virulence of Alternaria alternata and DNA polymorphism, as well as the virulence variation genetic basis of Alternaria alternata among different reproductive generations in a tobacco field, 6 Alternaria alternata isolates collected from the same tobacco field at different growth stage were tested with virulence determination and random amplified polymorphic DNA (RAPD) marker.The results showed that pathogen virulence increased with the increase of reproductive generations, the virulence variation was caused by the variation of pathogenetic DNA gene of pathogen.The tested isolates could be divided into 2 groups based upon the virulence tests on 3 tobacco cultivars or 3 groups according to the RAPD marker.Of the 105 RAPD marker generated by the tested isolates amplified with 12 random primers, 95.24% showed polymorphism.The results indicated that there existed genetic diversity among these isolates, and a coherence relationship between virulence polymorphism and DNA polymorphism, however, there existed some variations as well.