Litsea populifolia (Hemsl.) Gamble, a deciduous arbor of Lauraceae, has potential and extensive application in Traditional Chinese Medicine (TCM), cosmetics and edible essence. However, this species distributes in a narrow geographical region (restricted in Sichuan province, China), which seriously prohibits the exploitation and utilization of L. populifolia. Understanding the genetic diversity of populations among L. populifolia is urgently necessary and helpful for the future breeding and diversity conservation. In this study, the genetic diversity of 84 leaf samples, which were collected from 7 different regions in Mount emei area, was analyzed by genotyping-by-sequencing (GBS) technique. After the construction of GBS library using standard pipeline, we identified 48,795 SNP sites globally, 47.69% and 30.71% of which were located in intronic regions and intergenic regions, respectively. According to the phylogenetic tree, principal components and population structure analysis, seven subgroups of samples were clustered into three clusters. The average of Ho and He was 0.27218and 0.20635, respectively. As a result of ANOVA, the percentage of intra-group variation reached 96.28%, while inter-group variation was only 3.72%. These results indicated that regional divergence issued the main factor of genetic diversity among L. populifolia in Emei Mountain, and the different altitudes in high altitude area also affected the genetic diversity of intra-population. The high nucleotide diversity in the populations and the high percentage of intra-group variation illustrated that genetic variation of L. populifolia in Emei Mountain mainly exists in intra-population. The characteristics of dioecism and cross-pollination in L. populifolia caused higher gene flow. However, due to the fragmentation of its habitat and the complex vertical climate, there was a low gene flow between the groups, causing obvious population differentiation and the variation mainly within the populations. Our data provided a fundamental basis for the genetic breeding and diversity conservation of Litsea populifolia. In terms of family line selection, a large number of samples should be included in the population, and the preservation of germplasm resources should be at a lower altitude as far as possible.