Abstract: Sixty-six isolates were obtained through the isolation method of single spore from the infected plant tissues of tomato from 11 counties in Shandong Province. Morphologic and 5.8S rDNA-internal transcribed spacer (ITS) sequence analysis showed that the sixty-six isolates are all the Botrytis cinerea. In addition, all the ITS sequences of the sixty-six isolates were divided into two types through the sequence alignment, and only a single base differences between two types. Genetic diversity analysis of the 66 isolates was investigated by the inter-simple sequence repeats (ISSR). As a result, twenty-four sites were generated in total, among which 58.33% were polymorphic. The result indicated there was low genetic diversity among the Botrytis cinerea populations. By UPGMA cluster analysis, the isolates were classified into four subgroups and the clustering had relationships in association with the species and geographical origin.