Understanding epidemiology of the tick-borne pathogens requires the accurate identification of the vector ticks. Morphological analysis of ticks is difficult and often leads to misidentification. Molecular techniques offer an alternative approach of tick identification. To date, no practical and reliable molecular assays for discrimination of Euro-Asian ticks are available. Our aim was to develop such an assay for discrimination between four Euro-Asian tick species of high medical importance such as Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulatus. As a basis, we have chosen conventional species-specific polymerase chain reaction (PCR), a technique providing a good combination of simplicity and reliability. The DNA information available on ticks was searched for orthologous loci containing stretches of sequence dissimilarity sufficient for designing species-specific primers. ITS2 locus (second internal transcribed region of the rRNA gene cluster) was found to be the most favorable for primer design. Finally, for each of the three Ixodes species a PCR was developed amplifying only for the targeted species. One PCR amplified the entire ITS2 locus of the four species and allowed discrimination of D. reticulatus from the Ixodes species on the basis of the size difference of the respective PCR products. This PCR system was successfully tested for discrimination of the ticks at different maturation stages (larva, nymph, and adult) in engorged and unfed conditions, and therefore it may be useful for large-scale epidemiological studies. Differentiation between the closely related I. ricinus and I. persulcatus, the two species most often occurring in the tick-borne diseases in Eurasia, is of special importance.