Lychnis wilfordii (Regel) Maxim. is a perennial and narrowly distributed in Gangwon province of Korea. Also, it is rare and critically endangered which is subjected to strict protection as an endangered plant. Thus, population genetic study of this species is needed to prepare for management and conservation strategy. We developed microsatellite markers and used them to analyze genetic diversity of L. wilfordii populations. One hundred and forty five individuals from Korea (KI1, KI2, KP), China (CX, CF) and Russia (RP) were genotyped for 17 polymorphic microsatellite loci to assess genetic structure and variation of L. wilfordii populations. Korean popoulation had lower genetic diversity than others. Especially, KP population showed significantly lower observed heterozygosity, expected heterozygosity, Shannon's index (I) than KI1 and KI2 populations. Also, PCoA and STRUCTURE results showed that the KP population was isolated from all other populations. These aspects explained by effect from genetic drift. Discontinuous distribution leads to population isolation and fragmentation. Fragmentation of habitats has decreased genetic diversity within populations and increased genetic differentiation among populations. That, in turn, reduced their fitness and survival. To maintain high levels of genetic variation in species in fragmented habitats, conservation should be considered for securing connectivity between spatially distinct populations.