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Background: Linear dermatitis is endemic in Iran where most cases occur in the Caspian Sea coast and Fars province. The disease is caused by beetles of the genus Paederus which are active from early spring to beginning of autumn although its incidence rises from May to August. The classic taxonomy of Paederus spp. is based on the male genitalia that is very complex and needs expertise. In this study, we report a DNA-based method to discriminate Paederus fuscipes and Paederus littoralis (=syn: P. lenkoranus, P. ilsae). Methods: Type specimens were collected from north and south of Iran. Molecular typing of the species was performed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified fragments of mtDNA-COI. Results: Sequence analyses of the data obtained in this study showed significant DNA polymorphisms. There were 89 substitutions between COI sequences of the two species. The mtDNA-COI fragment comprises several useful species-specific restriction sites comprising HaeIII that could result in distinctively different species-specific PCR- RFLP profiles. The HaeIII enzyme cuts the 872 bp PCR amplicon of P. littoralis into 737 and 100 bp and two small nonvisible bands whereas it does not cut P. fuscipes amplicon into fragments. Conclusion: This study demonstrates that molecular typing is useful method and allows one to differentiate between two species and is recommended for discrimination of other Paederus species, which morphologically are indistinguishable or very difficult to be distinguished. [ABSTRACT FROM AUTHOR]