Abstract: In order to study the relationship between the isolate virulence and reproductive order of generation of Alternaria alternata Keissler, the outdoor inoculation tests in vivo and indoor tests in vitro were conducted to determine the virulence of six isolates of A.alternata from different growing stages of tobacco plants, and the virulence differences were compared with cluster analysis.The results showed that the virulence differences of the isolates were significant, the virulences of isolates No.4, 5 and 6 from late growing stage were stronger than those of isolates No.1, 2, and 3 from early stage, namely, the virulence of the virus becomes stronger generation after generation. The cluster dendrogram of virulence analysis gave the same results.The isolates could be divided into two groups based on their virulence reactions on three tobacco cultivars:one was isolated from early growing stage and another was from late stage.