Abstract:AimsTilia amurensis is one of the II-grade Chinese protected plants with an important economic value and ecological significance. The population of T. amurensis has gradually declined due to overdrafting. Our objectives were to 1) study the genetic diversity of six natural populations varying in longitude and latitude and six varying in altitude and 2) discuss strategies to conserve germplasm resources. Methods The newly developed inter-simple sequence repeats (ISSR) marker was used to examine genetic diversity. Important findings The results of 14 ISSR primers showed that polymeric percentage of the six populations was 93.85％, the gene diversity (H) was 0.243 3 and Shannon Index of diversity (I) was 0.380 3. The genetic diversities of T. amurens is decreased with increasing altitude, had a single-peak curve with increasing latitude, and were higher in the central region than in the edges. Analysis of molecular variance (AMOVA) revealed that the majority of genetic variation occurred within populations. Genetic distance and geographic distance were uncorrelated. We concluded that the genetic diversity of T. amurensis was high and mainly with in populations. Also, the major factors threatening the development of T. amurensis included direct damage to natural populations due to destruction of habitat and poor regeneration ability caused by innate biological and ecological characteristics. Habitat should be protected, as individuals, especially in core areas.