The Botrychium lunaria (Ophioglossaceae) complex worldwide includes the named species B. lunaria, B. crenulatum, B. tunux, and B. yaaxudakeit. These species have been distinguished from each other morphologically and genetically. This study further investigates the genetic diversity and geographic distribution of this complex, examining a large number of plants worldwide. Enzyme electrophoresis was used to examine allelic variation of 22 loci for 1574 plants of putative B. lunaria, B. crenulatum and B. tunux from North America, Eurasia, and New Zealand, and B. dusenii from the Falkland Islands. Variation in allelic composition assessed by genetic identity and cluster analysis using the programs PopGene and STRUCTURE as well as morphology and geography indicated that the complex is composed of six distinct entities; two of which warrant recognition as new species, B. neolunaria, endemic to North America, and B. nordicum, sister to the B. lunaria complex, from Iceland and Norway; and a new combination, B. lunaria var. melzeri, endemic to Greenland, Iceland, and Norway. The new taxa are described in this paper. Three entities within B. tunux are discussed but not proposed for recognition at this time. Botrychium lanceolatum, included in this study, is composed of three morphologically and genetically distinct entities warranting taxonomic recognition.