Karenia mikimotoi is a toxic dinoflagellate that is known to form extensive populations in the Eastern North Atlantic and Pacific Oceans that that impacts significantly on recreation and fisheries industries in these areas. Attempts to resolve the complex taxonomy within this ‘‘species’’ have been hampered by the requirement for fine-scale morphological analyses and by the lack of suitable genetic markers. Here we report the use of a novel combination of primer sets designed to facilitate amplification of the rDNA LSU and ITS, and the rbcL genes that can be used to discriminate between K. mikimotoi isolates originating from different geographical regions. We show that isolates from Europe and New Zealand are more closely related to each other than either is to isolates from Japan. Specific PCR-based primers were designed to amplify a region of the rbcL gene for subsequent high resolution analysis of the PCR amplicon melting temperatures. This innovative technique allows us to rapidly discriminate K. mikimotoi from distinct geographic localities and we propose the separation of K. mikimotoi into two distinct sub-species.