Ancient trees can serve as genetic reservoirs for special genes, and they are of significance in genetic applications dealing with forest fragmentation, climate change and drought. The Mausoleum of the Yellow Emperor in Shaanxi China has the largest number of ancient Platycladus orientalis trees. Here, Simple sequence repeat (SSR) technique was used to study the genetic diversity of ancient P. orientalis. Fifty-nine trees were sampled in four age groups and analyzed with 24 SSR markers. This set of SSRs had a moderate genetic diversity (mean He=0.327). Population structure and discriminant analysis of principal components (DAPC) both showed that these individuals were subdivided into four groups and the majority of the 19 oldest trees had a significant different genetic pool, compared to other age groups. This genetic differentiation could be caused by geographic or temporal originality. Our results show that ancient trees represent potential genetic resources for conservations. Better management of these ancient individuals and possible vegetative propagation could play an important role in the genetic conservation of these ancient trees.