Sceptridium dissectum, the most variable North American grapefern species, demonstrates variation in degree of blade dissection, blade color, pinnule shape, and pinnule margins. Historically, various morphologies have been included within S. dissectum. For example, Clausen's monograph recognized five infraspecific taxa in S. dissectum, of which only the morphologies of variety dissectum and obliquum are currently retained. However, the taxonomic status of the two varieties has been debated. We used ISSR (Inter-Simple Sequence Repeat) markers to assess the genetic distinctness of S. dissectum var. dissectum and var. obliquum in 17 Ohio populations. Five ISSR primers generated 69 reproducible loci. In UPGMA analyses and AMOVA, S. dissectum var. dissectum individuals did not cluster separately from var. obliquum individuals, nor did individuals from the same population cluster together. ISSR markers revealed levels of population genetic structure in S. dissectum similar to levels detected by previous isozyme investigations. Our results concur with recent treatments of S. dissectum that do not formally recognize infraspecific taxa, and may bring into question current species circumscriptions in Sceptridium. We illustrate the use of ISSR markers for examining taxonomic boundaries in Sceptridium.