Pteroceltis tatarinowii Maxim. (Ulmaceae), a tertiary relict plant of a temperate deciduous tree species endemic to China, is widely distributed in bare limestone mountains across the mainland in China. The bark (phloem fiber) of this plant has long since been used as the sole raw material for manufacturing Chinese traditional Xuan Paper. However, the species are subject to many threats due to its distribution pattern characterized by small patches; its decreasing population size resulted from overexploitation, and reduction of the original forest ecosystem. Thus, it has now been listed as a rare and endangered plant (National Grand III) in China. Using inter-simple sequence repeat markers, the genetic diversity and structure of 628 individuals from 27 populations of P. tatarinowii were detected. A total of 66 bands, of which 63 were polymorphic, were presented from the 8 selected primers screening across all samples, with the percentage of polymorphic bands up to 95.45%. The result of POPGENE revealed quite high level genetic diversity for the plant at the species level (PPB=95.45%,Ao=1.9545,Ae=1.5729,He=0.3335,I=0.4980). At the population level, TS population from Gansu harbored the highest genetic diversity (PPB=84.85%,Ao=1.8485,Ae=1.5217,He=0.3033,I=0.4516), whereas NL population from Guangdong with the lowest genetic variation (PPB=54.55%,Ao=1.5455,Ae=1.3135,He=0.1841,I=0.2756). The mean population genetic level (PPB=69.98%,Ao=1.6998,Ae=1.4449,He=0.2561,I=0.3793) and population genetic differentiation of P. tatarinowii (Gst=0.23,Ф_ST=0.25) were both at the middle level compared to other species. Gene flow (Nm) was estimated to be 1.65. In addition, different genetic variation and patterns were found between North China and South China. Populations of North China presented higher genetic diversity (PPB=95.45%, He=0.3332, I=0.4964) and lower genetic differentiation (Ф_ST=0.22) than those of South China (PPB=93.94%, He=0.3220, I=0.4814 and Ф_ST=0.25). It would seem the extant genetic pattern of P. tatarinowii was might mainly attributed to its long evolutionary history, wide-ranging distribution, outcross mating system, long life cycle and complex differences of terrain and vegetation between North China and South China. According to our aforementioned results and the evidence from our cpDNA data, in situ conservation was the preferred way to maintain the species' high level genetic diversity. Especially, much more attention should be paid to populations with higher genetic diversity (TS, JX and QY) and populations harboring peculiar cpDNA haplotypes (XA,SD,ML,NXS,LP,YF,WYS,GL,AL). In the condition of establishment of artificial plantation and germplasm bank, the above peculiar populations should been given prior consideration. Regarding the genetic pattern difference between South China and North China, more populations in South China and fewer representative populations with more individuals in North China should been sampled to obtain the utmost genetic diversity of P. tatarinowii.