BACKGROUND: This study was performed to investigate the phenotypic, karyomorphological, and habitat environment characteristics of Lilium distichum that grows naturally in South Korea. Currently, this species follows limited distribution areas and its natural populations are at the brink of extinction mainly due to fragmentation or destruction its natural habitat. RESULTS: This species was distributed between approximately 1,000 and 1,500 m above sea level with an average temperature of 22°C. The soil characteristics surrounding the natural habitats included loamy and silt loam soils having organic matter content (10.82%), pH (5.22), electrical conductivity (EC) (0.37 dS/m), total nitrogen (0.45%), and cation exchange capacity (34.3 cmol+/kg). The peak period of blossoming was between 27 July and 1 August. The maximum number of flowers was observed in Mount Deogyu (2.8), whereas the minimum number of flowers was observed in Mount Jiri (1.3) and Mount Seorak (1.2). Results regarding the number of verticillate leaf, bract counts, and verticillate leaf length and width were highest in Mount Odae, while lowest in the Mount Seorak region. The chromosome complement of L. distichum is 2n = 2x = 24; the length of somatic metaphase chromosomes ranges from 17.01 ± 0.32 μm (chromosome 10) to 32.06 ± 0.35 μm (chromosome 1) with a total length/genome of 261.92 μm. In L. distichum, the presence of 1 pair (two loci) of 5S ribosomal DNA (rDNA) and 8 pairs (16 loci) of 45S rDNA was revealed on metaphase chromosomes. One pair of 5S rDNA signal was observed in interstitial region of long arm of chromosome 3 which co-occurred with 45S rDNA. Among the eight pairs of 45SrDNA, three pairs of 45S rDNA signals were observed in short arm of chromosome (chromosome 2, 6, and 7) which were located close to centromere. The other five pairs of 45S rDNA signals were positioned in the interstitial region of long arm (chromosome 3, 4, 5, 10, and 11. CONCLUSIONS: This study provides baseline information regarding the effective exploitation and use of L. distichum resources for breeding research to be used as cut flower and potted plants.