Cimicifuga simplex Wormsk. (Ranunculaceae) is a perennial herb distributed in eastern and northeastern Asia for which at least three different pollination morphs have been reported. It is classified as endangered or near threatened in some Japanese regions, and its rhizome is commercially used as a crude drug. To examine genetic differentiation and gene flow among the three morphs, we developed eight microsatellite markers by using next-generation sequencing and estimated the genetic structure of C. simplex. We tested eight primer pairs on 93 individuals from six populations of C. simplex in Nagano, central Japan, and found that heterozygosity in morphs I and III was low compared to expected heterozygosity. Bayesian clustering performed with the STRUCTURE program clearly distinguished the three morphs of C. simplex, and only a little gene flow was detected among the morphs. These eight microsatellite markers are expected to be useful in conservation genetic studies of this species and for future conservation planning.