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  The genus Scylla, recently divided into four separate species: S. paramamosain, S. serrata, S.tranquebarica and S. olivacea, is a set of large and primarily carnivorous portunid crabs. Morphologicalapproaches are difficult to identify the four species from each other unambiguously, especially in thosecases where morphological features are missing or misleading. DNA barcoding has been considered tobe an effective standardized tool for identification of animal species within a known taxonomicframework. In the present study, we examined the utility of COI sequences as DNA barcoding foreffective identification of four species of genus Scylla. The average intraspecific K2P distances were0.40% in S. paramamosain, 1.4% in S. serrata, 1.5% in S. olivacea and 0.6% in S. tranquebarica,respectively. The interspecific K2P distances were much higher than the intraspecific distances, and themaximum interspecific distance (19.8%) was between S. paramamosain and S. olivacea, while theminimum interspecific distance (9.2%) was between S. paramamosain and S. tranquebarica. Nooverlaps were detected present between intraspecific and interspecific distances of four species,suggesting the existence of distinct barcoding gaps. The neighbour-joining (NJ) tree consisted of fourdistinct clusters, with all sequences of each species forming a strong monophyletic cluster. Furthermore,we developed a PCR-based species identification method that could identify the four species of genusScylla rapidly and effectively. This paper provided two methods including DNA barcoding andPCR-based protocols for species identification of genus Scylla, that will be useful for linking differentdevelopmental stages and hybridization breeding program of genus Scylla.……