In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238-1819 bp, and the lengths of ITS-2 were 137-1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0-1.5%, nucleotide differences between hard tick species ranged 2-25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks.