Athyrium filix-femina (Lady Fern) comprises a complex of homoploid (n = 40) taxa, distributed over much of the northern hemisphere and extending into South America, whose evolutionary relationships are poorly understood and whose taxonomic treatment is problematic. The A. filix-femina complex of North America comprises as many as four taxa with overlapping ranges and provides an especially suitable context for exploring patterns and processes of divergent evolution and its taxonomic consequences in ferns. We addressed differentiation of two eastern North American taxa distinguished on the basis of growth form, frond shape, and spore color, and most recently treated as varieties A. angustum and A. asplenioides (Northern and Southern Lady Fern respectively). Although, these two taxa have been long perceived as closely related, they have been known to intergrade and recombine to form a hybrid zone in their relatively narrow region of overlap. This perception is supported by the data from the present study. Collections from 17 populations, 9 of A. angustum from Quebec to Pennsylvania and 8 of A. asplenioides from New Jersey to North Carolina, were examined for spores using LM, SEM, and TEM, and/or allozymes (16 loci coding 10 enzymes). The two taxa exhibited highly distinct perispore surfaces: all A. angustum individuals had papillose surfaces, whereas most A. asplenioides individuals were rugose with a reticulum of inflated folds. Spores from the northernmost A. asplenioides population sampled (Shirley, NJ) showed varying degrees of intermediacy suggestive of introgressive hybridization with A. angustum. Levels of allozyme polymorphism in populations (means: P=36.5%, A=1.97, HE=0.129) were near means for angiosperms and ferns. Genotype frequencies at most loci in all populations were in Hardy-Weinberg equilibrium indicating an outcrossing mating system. Most alleles were shared among all populations. However, at the four most polymorphic loci (Idh-1, Pgi-2, Pgm-2, and Tpi-2) allele frequencies were significantly divergent between populations of A. angustum and A. asplenioides, especially Idh-1 which approached fixation for alternate alleles. Values for FST ranged from 0.008 to 0.459 for individual loci (0.255 across loci) with especially high values for Idh-1, Pgi-2, Pgm-2, and Tpi-2. Hierarchical FST analysis indicated that differences between the two taxa (FXY=0.216) accounted for most allele frequency divergence among populations (FXY=0.238). UPGMA analysis of paired Rogers' Similarity (S) values resulted in two principal clusters each comprising populations of one taxon. Populations of A. angustum and A. asplenioides were joined within their clusters at S=0.938 and S=0.945 respectively, while the two taxon clusters were joined at S=0.848. The spore and isozyme data indicate substantial divergence between A. angustum and A. asplenioides, suggesting that they merit distinction at the rank of subspecies or species. Additional study of populations in their region of sympatry is required to determine the nature and extent of hybridization.