Technical Abstract: Two biotypes of Hydrilla verticillata occur in the United States, a dioecious type centered in the southeast and a monoecious type in the central Atlantic and northeastern states. Ecosystem managers need tools to distinguish the types as the ranges of each type expand and begin to overlap. A molecular tool using RAPDs is available but its use is limited by a need for reference samples. We describe an alternative molecular tool which uses 'universal primers' to sequence the trnL intron and trnL-F intergenic spacer of the chloroplast genome. This sequence yields three differences between the biotypes (two gaps and one single nucleotide polymorphism). A primer has been designed which ends in a gap that shows up only in the dioecious plant. PCR using this primer produces a product for the monoecious but not the dioecious plant.