Biological species are traditionally identified based on their morphological features and the correct identification of species is critical in biological studies. However, some plant types, such as seagrass, are taxonomically problematic and difficult to identify. Furthermore, closely related seagrass species, such as Halophila spp, form a taxonomically unresolved complex. Although some seagrass taxa are easy to recognize, most species are difficult to identify without skilled taxonomic or molecular techniques. Barcoding coupled with High Resolution Melting analysis (BAR-HRM) offers a potentially reliable, rapid, and cost-effective method to confirm species. Here, DNA information of two chloroplast loci was used in combination with HRM analysis to discriminate four species of seagrass collected off the southern coast of Thailand. A distinct melting curve presenting one inflection point was generated for each species using rbcL primers. While the melting profiles of Cymodocea rotundata and Cymodocea serrulata were not statistically different, analysis of the normalized HRM curves produced with the rpoC primers allowed for their discrimination. The Bar-HRM technique showed promise in discriminating seagrass species and with further adaptations and improvements, could make for an effective and power tool for confirming seagrass species.