Abstract: In order to research the changes of genomic sequence after Stevia rebaudiana Bertoni diploid chromosome doubling, ISSR molecular marker technique was used to analyze the genetic variation between 1 diploids and 5 autotetraploids of Stevia rebaudiana Bertoni. 5 primers with good polymorphic and steady bands were selected from 60 ISSR primers, and 34 bands were amplified totally. 19 of 34 bands were polymorphic, the polymorphic rate was 55.88%. The genetic diversity index were 0.0059-0.2778 in 6 Stevia rebaudiana Bertoni lines. The UPGMA dendrogram showed that CK and 5 tetraploids separated firstly, which explaining that Stevia rebaudiana Bertoni diploids and autotetraploids were separated well by cluster analysis. 1 and 2 lines, 4 and 5 lines clustered together firstly, which indicating that the genetic similarity coefficient of the two groups were high. There was significant genetic variation between autotetraploid and diploid of Stevia rebaudiana Bertoni, and different lines with the same ploidy also had genetic variation.