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Genetic diversity and population differentiation of the blue swimming crab (Portunus pelagicus) in Thailand, originating from Ranong and Krabi located in the Andaman Sea (west) and Chanthaburi, Prachuap Khiri Khan, and Suratthani located in the Gulf of Thailand (east), were examined by AFLP analysis. High genetic diversity of P. pelagicus in Thai waters was found (N = 72). The four primer combinations generated 227 AFLP fragments, and the percentage of polymorphic bands in each geographic sample was 66.19–94.38%. The mean genetic distance between pairs of samples was 0.1151–0.2440. Geographic heterogeneity analyses using the exact test and FST-based statistics between all pairwise comparisons were statistically significant (P < 0.01), indicating a fine-scale level of intraspecific population differentiation of Thai P. pelagicus. The estimated number of migrants per generation (Nem) was 0.26–0.76, suggesting restricted gene flow levels of P. pelagicus in Thai waters.