Abstract: This work is to further reveal the molecular mechanism of the biosynthesis of steviol glycosides（SGs）and analyze the reasons caused the different accumulation of SGs in stevia leaves. Different stevia materials were utilized to clone the coding genes of kaurenoic acid hydroxylase（KAH）at the key point of a branch pathway，and the biological function of the obtained nucleic acid sequences were analyzed and predicted. Finally six highly homologous nucleotide sequences were cloned. The key base mutations of nucleotide sequences caused significant differences in the location and size of the open reading frame（ORF），and totally 7 different KAH genes were identified. Both KAH1 and KAH2 were found in SR1，SR3，Xinfeng 3，Puxing 1 and Shoutian 3，KAH3 was cloned in Jiangtian 1 and Shoutian 3，KAH4 were found in Jiangtian 3，and KAH5，KAH6 and KAH7 all existed in SR2. Furthermore，SrKAHs showed the highest expression in stevia varieties and tissues with high accumulation of SGs. P450 conserved domains were in the all of them，and further evolution analysis and amino acid homology results showed that they belonged to the CYP716 family. And the predicted results of subcellular location showed KAH2 and KAH4 localized in cytoplasm while the other five were localized in the chloroplast. Differences of gene numbers，amino acid length and location in subcellular structures of KAH genes in Stevia maybe cause the different expressions and functions of SrKAHs，finally resulting the different accumulation of SGs.

Key words: Stevia rebaudiana, steviol glycosides, KAH, CYP716