In this study, we developed a simple and rapid restriction fragment length polymorphism (RFLP) method to distinguish the sibling species Hemigrapsus penicillatus and H. takanoi based on interspecific base substitutions within the 16S rRNA region of mitochondrial DNA. Sequencing and alignment of partial sequences revealed two haplotypes for H. penicillatus and one for H. takanoi, and five species-specific base substitutions. Digestion of the PCR products using the restriction endonuclease Dde I produced a banding pattern that was consistent with species identification based on morphological and pigmentation patterns. The sequence for H. takanoi collected in Japan showed high homology with that reported for specimens collected in Europe. This method could be useful for identification of these two species by researchers without specific taxonomic knowledge.