Pistacia chinensis Bunge (Anacardiaceae) is a dioecious woody plant of significant economic values that is used in traditional Chinese Medicine as well as for wood production. More importantly, it is one of the ideal tree species for bio-diesel production because of the high oil content in its seeds. In this study, we aim to reveal the effects of landscape fragmentation on the genetic diversity (GD) of the dioecious plant Pistacia chinensis populations. A total of nine microsatellites were used to genotype 180 P. chinensis individuals from six populations to estimate the differences in GD between different populations. The study revealed that genetic diversity of the P. chinensis population as a whole is relatively high in the Thousand-Island Lake (TIL) region, but its fragmented landscape still led to the loss of rare alleles, especially in a fragmented small population, a post-fragmented population, and a male population. The partitioning of a large continuous population into small isolated remnant patches led to the direct loss of genetic diversity and, subsequently, because of the mediated gene flow of seeds and pollen, genetic drift, and the spatial distribution of existing plants, the GD gradually decreased. The restricted gene flow and the increase in self-pollination and inbreeding impaired the population’s long-term development. Therefore, the wild P. chinensis populations in the TIL region needs effective protective measures, including foreign artificial pollination and seedling transplantations.