A presumed hybrid mud crab of the genus Scylla was examined. The presumed hybrid could not be easily separated morphologically from the three Japanese Scylla species, S. serrata, S. paramamosain, and S. olivacea. An analysis of the first internal transcribed spacer (ITS-1) of the nuclear DNA revealed that the hybrid carried genomic DNA from both S. serrata and S. olivacea. Analysis of the maternally inherited 16S rDNA of the mitochondrial DNA demonstrated that the presumed hybrid carried S. olivacea mtDNA. Based on these results, we conclude that the examined animal is indeed the hybrid offspring of a female S. olivacea and a male S. serrata. The degree of genetic isolation among these species, and the possible causes of hybridization are discussed.