ABSTRACT The phylogeny of closely related species of Isoetes has been difficult to infer using morphological or molecular data. The southeastern United States exhibits among the highest diversity of Isoetes species, which likely are the result of a relatively recent radiation. We used whole chloroplast genome (plastome) sequences to infer a phylogeny of diploid taxa in the Southeast. The phylogeny of the entire plastome was well supported across various models of evolution. Separate phylogenetic analyses of coding regions and introns mostly returned the same tree topology as the whole plastome, although the placement of four species was discordant. The plastome phylogeny highlights taxonomic issues, because two pairs of infrageneric taxa are resolved as polyphyletic. Inference of ancestral character states suggests a common ancestor of the southeastern clade with white, tuberculate megaspores and papillose microspores maturing in the spring. This study is the first to resolve molecular-phylogenetic relationships in diploid Isoetes from the southeastern USA and shows the utility of plastome sequences for unraveling relationships between closely related taxa.