Phylogenetic relationships among 23 species of Botrychium s. s. were investigated using sequence data from three plastid DNA regions: rbcL, trnL^UAA -trnF^GAA intergenic spacer, and rpL16 intron. Individual and combined maximum parsimony and a combined maximum likelihood analysis showed strong support for the monophyly of Botrychium s. s., and all analyses identified three main, well-supported clades. However, within those three clades all analyses did not produce identical tree topologies. The Lanceolatum clade contained the diploid B. lanceolatum and seven polyploids, the Lunaria clade contained the diploids B. lunaria and B. crenulatum, and the Simplex-Campestre clade contained 13 species distributed across five subclades. Relationships among the five subclades were not resolved in either of the combined analyses. The Simplex subclade contained four diploids (B. simplex, B. montanum, B. mormo, and B. pumicola), the Pallidum subclade contained the diploid B. pallidum and the tetraploid B. gallicomontanum, the Minganense subclade contained three tetraploids (B. minganense, B. paradoxum, and B. × watertonense), the Ascendens subclade contained the tetraploid B. ascendens, and the Campestre subclade contained the diploids B. campestre and B. lineare and the tetraploid B. spathulatum. Multiple accessions of five species revealed cpDNA sequence variation within four species, and potential species or population-specific substitutions were identified for some taxa.