In order to explore the relationship between the accumulation of steviol glycosides (SGs) and expression of SGs biosynthesis pathway genes, SGs content and composition in stevia leaves of different canopies of one variety (xinfeng 3) and three varieties (shoutian 3, jiangtian3, puxing 1) were analyzed and the related genes expression were tested by real-time PCR. The results showed that the accumulation of total SGs was highest in the top leaves and lowest in the bottom leaves of xinfeng 3. Among the eight tested genes, six of them had the highest expression in the top leaves and had the lowest in the bottom leaves. The stevioside accumulation was highest in top leaves and the expression of its catalytic enzyme encoding gene SrUGT74G1 was also highest in top leaves. And rebaudioside A (RA) content was the highest in bottom leaves and the expression of its catalytic enzyme encoding gene SrUGT76G1 was highest in the bottom leaves. Among three varieties, the accumulation of total SGs and rebaudioside A (RA) was highest in jiangtian 3 and lowest in puxing 1, while the stevioside accumulation was highest in shoutian 3 and lowest in jiangtian 3. However expression of related genes did not show the same trend to SGs accumulation in three stevia varieties. So it was deduced that expression level of SGs biosynthesis pathway genes affected the SGs accumulation significantly, and in the same variety, the expression level of catalytic enzyme encoding gene could reflect the accumulation of corresponding steviol glycoside.