Microsatellite markers were isolated for Daphne kiusiana var. kiusiana (Thymelaeaceae), an evergreen broad-leaved shrub endemic to Korea and Japan. Because its populations in Jeju Island are morphologically controversial, and consistently threatened by anthropogenic pressures, taxonomic delimitation and conservation effort are required at the genetic level. We developed 21 polymorphic microsatellite loci from Next Generation Sequencing data. The primer set included di-, tri-, and tetra-nucleotide repeats. Variability in the markers was tested for 80 individuals of D. kiusiana from three natural populations in Jeju Island and Japan. Among the 21 loci, three were unavailable for population JKJU of Japan. The Neighbor-Joining tree based on microsatellite markers described here classified the three populations into two groups according to geographical or morphological traits. These will be a powerful genetics tool for determining the taxonomic boundary and establishing suitable conservation strategies for D. kiusiana in Jeju Island.