Abstract: In order to systematically analyze the genetic diversity pattern of Perilla frutescens germplasm resources in China, 70 representative germplasm resources from 12 provinces covering the northern and southern regions were selected. The genetic diversity, population structure and genetic relationship of 70 Perilla frutescens were systematically analyzed by using Hyper-seq simplified genome sequencing technology to carry out the genome level scanning. The results showed that: a total of 1 918 357 high-quality single nucleotide polymorphism (SNPs) loci and 766 430 insertion-deletion (InDel) markers were identified on 20 chromosomes of Perilla frutescens by mutation detection and annotation. On average, about 95 918 SNPs and 38 322 InDels were detected on each chromosome. The variation was mainly distributed in the intergenic spacer region (69.16%), and 237 564 variation sites were identified in the exon region. The results of Neighbor-joining tree, principal component analysis (PCA) and Admixture analysis based on SNP diversity consistently showed that: the 70 Perilla frutescens germplasm resources could be divided into four major genetic subgroups, which were the Shanxi-Inner Mongolia-Liaoning inland group (n=25), the Liaodong coastal and eastern mountainous group (n=13), the Heilongjiang group (n=8) and the Central-Southwest China group (n=24). The population structure was highly correlated with the geographical origin. The population genetic diversity assessment showed that the overall Nei’s index was approximately 0.63, and the nucleotide diversity (Pi) was 0.39 and 0.34 between the core collection and the reserve collection. The observed heterozygosity (Ho) was slightly lower than the expected heterozygosity (He), suggesting that there were structural differences in allele frequencies among populations. The core collection of 18 accessions screened based on Genocore could cover over 90% of the total genotypic variation. The results revealed the regional differentiation and diversity pattern of Perilla frutescens germplasm, and identified two novel genetic subgroups of Liaodong coastal and eastern mountain group and Heilongjiang group. The unique genetic status of Perilla frutescens in Yunnan was clarified, which provided a genomic basis for germplasm development, molecular marker-assisted identification and resource utilization of Perilla frutescens in different regions, and was of great significance for promoting the sustainable development of Perilla frutescens industry and the protection of germplasm resources diversity.