BackgroundGenetic diversity, genetic structure, and gene flow in plant populations and their influencing factors are important in conservation biology.Cypripedium macranthosis one of the few wild orchids with high ornamental value in northern China. However, over the past decade, excessive collection, trading, tourism development, habitat fragmentation, deceptive pollination, and seed germination difficulties have all caused a sharp decline in the number ofC. macranthosindividuals and its population. In order to propose a scientific and effective conservation strategy, the genetic diversity, genetic structure and gene flow of the current CM population are urgent scientific issues to be clarified.ResultsHere, 99 individuals ofC. macranthosfrom north and northeast China were analyzed to evaluate the genetic diversity, gene flow among populations, and genetic structure by genotyping-by-sequencing. More than 68.44 Gb high-quality clean reads and 41,154 SNPs were obtained. Our data based on bioinformatics methods revealed thatC. macranthoshas lower genetic diversity, high levels of historical gene flow, and moderate-to-high genetic differentiation between populations. The gene migration model revealed that the direction of gene flow was mainly from northeast populations to north populations in China. The results of genetic structure analysis showed that 11C. macranthospopulations can be considered as two groups, and further divided into four subgroups. Moreover, the Mantel test detected no significant “Isolation by Distance” between populations.ConclusionsOur study demonstrates that the present genetic diversity and genetic structure ofC. macranthospopulations were mainly caused by biological characteristics, human interference, habitat fragmentation, and restricted gene flow. Finally, constructive measures, which can provide a basis for the proposal of conservation strategies, have been suggested.