The first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA and mitochondrial DNA 16S rRNA were amplified by Polymerase chain reaction (PCR) using genomic DNA extracted from adult tissue of four species of Scylla spp. and the first zoeal stages of S. serrata, S. paramamosain and S. olivacea as template. Using the ITS-1 region, variation in product fragment length was found to be useful for distinguishing S. serrata and S. olivacea from two other species. The other two species (S. paramamosain and S. tranquebarica) could be identified using the restriction endonuclease Hha I. Using 16S rDNA, all four species were identified using PCR-restriction fragment length polymorphism (RFLP) by double digestion with DraI and HindIII. These genetic markers can be used for hybridization breeding studies and in field studies of larval and juvenile mud crabs of the genus Scylla.