Octopus vulgaris, Octopus cyanea and Callistoctopus ornatus may co-occur in near-shore shallow reef habitats across the Southwestern Indian Ocean. All three species are harvested, both by small vessels and individual spearfishers and are of considerable socio-economic importance. Phenotypic-based discrimination of the three species is unreliable and at present species-specific catch data are not collected, preventing evaluation of species specific fisheries effects. In light of increasing exploitation rates the development of an unambiguous means of species discrimination is required for sustainable management and conservation of species and intraspecific stocks. In this study, a simple, cost effective, molecular technique to differentiate the three species is described.