Ottelia acuminate (Gagnep.) Dandy is an aquatic plant species that is endemic to the Yunnan-Guizhou Plateau (YGP) and adjacent areas of southwestern China. The taxonomic status of this species is still controversial and the main debate is on the distinction of the varieties. To determine the extent of genetic divergence among the varieties of this species, we studied the phylogenetic relationship and divergence time among representative plants of previously recorded varieties. Our sampling included 169 individuals representing five recorded varieties from 11 populations. We sequenced five chloroplast DNA (cpDNA) non-coding regions and nuclear ITS region. We identified a total of 20 different cpDNA haplotypes and seven ITS ribotypes. Phylogenetic analyses revealed that cpDNA haplotypes from each variety except for O. acuminate var. jingxiensis formed a distinct lineage but only two ITS ribotype lineages were grouped by variety. Our results suggested that most of the varieties described based on morphological characters represent genetically differentiated entities. The divergence times between varieties were estimated to be at the late Tertiary and early Quaternary. Moreover, the genetic differentiations among O. acuminate populations (or varieties) might result from the limited gene flow among isolated populations in different aquatic environments, which were probably formed during the Pliocene and Pleistocene geologic movements of the YGP.