Phylogeny of the family Ophioglossaceae and a phylogenetic position of Mankyua were estimated through analyses of chloroplast rbcL gene sequences and spore morphology. Sequence analysis of the rbcL gene clearly indicated that there are two major lineages in the family Ophioglossaceae: Botrychioid lineage and Ophioglossoid lineage. The Botrichioid lineage is composed of three distinct clades: Botrychium, Helminthostachys and Mankyua, where Helminthostachys and Mankyua were placed as sister groups to the Botrychium. Within the genus Botrychium, subgenera Septridium and Botrychium were monophyletic, while taxa of subgen. Botrypus branched as sister of the two, successively, thus making a non-monophyletic group. Ophioglossum formed the Ophioglossoied lineage, where the subgen. Ophioglossum is monophyletic, while subgen. Cheiroglossa and Ophoderma formed a sister relationship with subgen. Ophioglossum. In terms of external morphology and spores, Mankyua is most similar to Helminthostachys, however, patristic distance in the cladogram and trophophore characteristics of the two genera are distinct. Therefore, Mankyua is a well defined genus within the family in terms of morphology as well as molecular phylogeny which places it in basal position of the Botrychioid lineage on the gene tree.