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Reliable and accurate species identification is essential to establish a strategy for monitoring and manipulation of plant populations for conservation biology. However, identification of nonflowering plants is difficult. Based on recent advances in molecular biological techniques, various molecular identification methodologies have been proposed and applied in plant science, with a focus on medicinally valuable species. Lady’s slipper orchids (Cypripedium) are a representative terrestrial orchid group that includes many endangered species. Of 46 species, 3 (Cypripedium japonicum Thunb., Cypripedium macranthos Sw., and Cypripedium guttatum var. koreanum Nakai) were reported from Korea and categorised as endangered species or at risk of becoming endangered because of the rapidly decreasing population. We generated an accurate molecular identification system for these species using the sequence variation and species-specific SNPs of two plastid loci, rpoC2 and the IGS region between atpF and atpH. We selected the atpF-H region for molecular identification of the Korean Cypripedium and related Taiwan-endemic taxon Cypripedium formosanum Hayata. Cypripedium guttatum var. koreanum contained large deletions of over 300 bp, and could be distinguished by electrophoresis. For the other three species, we designed new primers based on a specific insertion (C. macranthos) and SNPs (C. japonicum and C. formosanum). We confirmed that molecular identification enables detection of each species using species-specific primers.