The population genetics of ferns, which results from initiation of individuals in a new location (often via long-distance dispersal) plus a wide range of mating systems, merit continued study. In the case of species in the subfamily Botrychioideae (specifically the genera Botrychium and Botrypus), previous work using allozyme and isozyme techniques revealed low genetic diversity and weak population genetic structure. This lack of genetic differentiation between populations is in spite of underground fertilization in the genus resulting in high levels of inbreeding and primarily fixed heterozygosity in tetraploids. In the present study, Amplified Fragment-Length Polymorphisms (AFLPs) were used to examine population genetics and structure of three species in the genus Botrychium and one species in the genus Botrypus. Measures of population genetic diversity were generally low, with the highest measures in the relatively common Botrypus virginianus. Across all species, measures of population differentiation were low and most genetic variation was contained within populations. Bayesian analysis of population structure using the program STRUCTURE corroborated these findings, with inferred genetic clusters that generally did not correspond to geographic collecting locations. These results agree with previous studies, with low genetic diversity within and among populations likely due to self-fertilization that limits outcrossing and long-distance spore dispersal that results in genetically similar populations.