Geranium soboliferum var. kiusianum and var. hakusanense (Geraniaceae) are believed to be relict plants that migrated southward from the Eurasian continent to the Japanese archipelago during the last glacial period. G. soboliferum var. kiusianum individuals and populations, which occur in wetlands in the Aso-Kuju region of Kyushu Island, are declining as a result of the deteriorating quality of the microhabitat, a process that is associated with the decrease in semi-natural grasslands. Using 10 polymorphic microsatellite markers, we evaluated the genetic diversity and population structure of the G. soboliferum complex, with the aim of understanding the species’ genetic characteristics to facilitate efficient conservation. Genetic demographic analysis suggested that Japanese G. soboliferum populations branched off from the Eurasian continental G. soboliferum var. soboliferum during the last glacial period and that, thereafter, the Japanese populations of the plant diverged into two lineages at the beginning of the Holocene epoch. The genetic diversity of G. soboliferum var. kiusianum was significantly lower than that of var. hakusanense , which is more widely distributed in central Honshu Island (mean Ar  = 3.288 vs. 3.830 and H e = 0.366 vs. 0.546). Genetic differentiation among populations was significantly higher (mean F ST = 0.368 vs. 0.184) in var. kiusianum populations and, notably, exhibited non-significant patterns of isolation by distance, indicating that the populations underwent strong genetic drift independent of adjacent populations. Our findings suggested that the population’s isolation, and its associated habitat specialization and limited inter-population gene flow, would have accelerated genetic differentiation in var. kiusianum . Overall, G. soboliferum var. kiusianum , which is genetically unique to wetland habitats in the Aso-Kuju region, merits conservation, and appropriate human intervention and management are critical to maintaining its remaining habitats in semi-natural grasslands.