It is generally believed that the members of Ophioglossaceae have subterranean, potentially bisexual gametophytes, which favor intragametophytic selfing. In Ophioglossaceae, previous allozyme studies revealed substantial inbreeding within Botrychium species and Mankyua chejuense. However, little is known about the mating system in species of the genus Ophioglossum. Molecular marker analyses can provide insights into the relative occurrence of selfing versus cross-fertilization in the species of Ophioglossum. We investigated allozyme variation in 8 Korean populations of the homosporous fern Ophioglossum vulgatum to infer its mating system and to get some insight into the population-establishment history in South Korea. We detected homozygous genotypes for alternative alleles at several loci, which suggest the occurrence of intragametophytic self-fertilization. Populations harbor low within-population variation (% P = 7.2, A = 1.08, and H (e) = 0.026) and a high among-population differentiation (F (ST) = 0.733). This, together with the finding that alternative alleles were fixed at several loci, suggests that the number and size of populations of O. vulgatum might have been severely reduced during the last glaciation (i.e., due to its in situ persistence in small, isolated refugia). The combined effects of severe random genetic drift and high rates of intragametophytic selfing are likely responsible for the genetic structure displayed by this homosporous fern. Its low levels of genetic diversity in South Korea justify the implementation of some conservation measures to ensure its long-term preservation.