Phloem-feeding planthoppers of the genus Prokelisia rank among the most abundant and ecologically important browsers of coastal saltmarsh grasses of eastern North America and the Caribbean. Along the Spartina marshes of the northern Gulf of Mexico, the sympatric species P. marginata and P. dolus are the most abundant, but are difficult to distinguish from each other based solely on morphology. This study seeks to design a molecular assay based on High Resolution Melting Analysis (HRMA) as a fast, cost-effective alternative to differentiate these species. A 450 base pairs (bp) segment of cytochrome c oxidase subunit I (COI) was amplified and sequenced for representative samples of both species, and a short amplicon (SA) HRMA was designed based on the presence of fixed nucleotide differences between species found along a 60 bp segment of COI. The unambiguous identification of individual specimens of P. marginata or P. dolus was possible due to easily discernable differences in the melting temperatures of the two species along this mini barcode. This assay may prove useful for future genetic studies involving these species by preventing the overestimation of genetic diversity via inclusion of conspecifics, and in ecological studies by improving data on the effects of individual species of Prokelisia.