To clarify the genetic status and provide effective information for the conservation of Athyrium viridescentipes, a critically endangered fern species with only 103 individuals remaining in the wild, we conducted ubiquitous genotyping to determine the genotypes of all remnant individuals of the target species. We analyzed the genetic variation of the 103 known individuals in four populations by using 13 microsatellite loci. The genotypes of single spores from a sporophytic individual were also determined in order to reveal the breeding system of this species. The level of allelic variation in A. viridescentipes was significantly lower than that of closely related Athyrium species. The genetic composition of the four populations was rather similar. Sixty-nine individuals (67%) possessed an identical pattern in the allele combinations at 13 microsatellite loci. The mean pairwise F (ST) among four populations was 0.018. The segregated pattern of alleles, determined by single-spore genotyping, revealed that allelic recombination occurs through meiosis. The results indicate that this species contains a low level of genetic variation, has low population differentiation, and maintains populations by sexual reproduction. These findings could lead to more effective conservation programs, the selection of the most appropriate individuals for ex situ conservation efforts, and separate management of extant populations.