The genus Isoetes is known for morphological convergence and a relative paucity of useful gross morphological characters for identification. Thus, the chromosome complements of Isoetes spp. are key attributes for species delimitation. Like many other plant lineages, polyploidy and reticulate evolution are important drivers of speciation in Isoetes. In the southeastern US Isoetes flora, polyploidy is common. We used a rapid DNA flow cytometry protocol to generate C values for four diploid, one tetraploid, and one hexaploid Isoetes species and conducted preliminary cytological investigations of two communities of Isoetes. Our work showed noteworthy variation in diploid Isoetes 1C values (1.39 to 3.32), including the smallest 1C value reported for the genus, Isoetes melanopoda ssp. silvatica. The hexaploid species, Isoetes microvela had the highest 1C value (4.21), and the tetraploid species evaluated had an intermediate 1C value (3.37). However, based on the variation in diploid 1C values measured, a simple 2×, 4×, 6× ratio of C values was not apparent for diploids, tetraploids, and hexaploids, respectively. All Isoetes taxa measured had significantly different 1C values and homogenous C values were observed in two communities of Isoetes (n = 24). These results indicate that DNA flow cytometry may prove a useful tool for routine Isoetes species identification, systematic work, and population-level surveys of Isoetes ploidy level.