The genus Tanakaea is a plant genus that consists of one or two evergreen herbaceous species in Japan and China. As rithophytic plant species occur on shaded rocks, the populations are usually isolated and sporadically found in disjunct areas. To evaluate the genetic structure of the species at multiple spatial scales, 10 nuclear and mitochondrial microsatellite markers were developed. The novel markers showed high genetic variations (two to 15 alleles and He from 0.400 to 0.894). Clonal samples were identified with the probability of identity of 9.0E‐8. When evaluated with 11 populations in Japan, significant genetic differentiation between regional population groups was detected (FST = 0.313 between Shikoku and Honshu islands), suggesting they have long been isolated from each other. Overall, these markers will be useful for population genetic research to investigate clonal structure and genetic diversity and levels of genetic differentiation between the geographically isolated populations.