The genus Thujopsis (Cupressaceae) comprises monoecious coniferous trees endemic to Japan. This genus includes two varieties: Thujopsis dolabrata (L.f.) Siebold et Zucc. var. dolabrata (southern variety, Td) and Thujopsis dolabrata (L.f.) Siebold et Zucc. var. hondae Makino (northern variety, Th). The aim of this study is to understand the phylogeographic and genetic population relationships of the genus Thujopsis for the conservation of genetic resources and future breeding. A total of 609 trees from 22 populations were sampled, including six populations from the Td distribution range and 16 populations from the Th distribution range. The genotyping results for 19 expressed sequence tag (EST)-based simple sequence repeat (SSR) markers, followed by a structure analysis, neighbor-joining tree creation, an analysis of molecular variance (AMOVA), and hierarchical F statistics, supported the existence of two genetic clusters related to the distribution regions of the Td and Th varieties. The two variants, Td and Th, could be defined by their provenance, in spite of the ambiguous morphological differences between the varieties. The distribution ranges of both variants, which have been defined from their morphology, was confirmed by genetic analysis. The Th populations exhibited relatively uniform genetic diversity, most likely because Th refugia in the glacial period were scattered throughout their current distribution area. On the other hand, there was a tendency for Td’s genetic diversity to decrease from central to southern Honshu island. Notably, the structure analysis and neighbor-joining tree suggest the hybridization of the two varieties in the contact zone. More detailed studies of the genetic structure of Td are required in future analyses.