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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. Morphological approaches are difficult to identify the four species from each other unambiguously, especially in those cases where morphological features are missing or misleading. DNA barcoding has been considered to be an effective standardized tool for identification of animal species within a known taxonomic framework. In the present study, we examined the utility of COI sequences as DNA barcoding for effective identification of four species of genus Scylla. The average intraspecific K2P distances were 0.40[％] 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 the maximum interspecific distance（19.8[％]）was between S. paramamosain and S. olivacea, while the minimum interspecific distance（9.2[％]）was between S. paramamosain and S. tranquebarica. No overlaps 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 four distinct 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 genus Scylla rapidly and effectively. This paper provided two methods including DNA barcoding and PCR-based protocols for species identification of genus Scylla, that will be useful for linking different developmental stages and hybridization breeding program of genus Scylla.