Abstract Objectives: Hard ticks (Acari: Ixodidae) are ectoparasites of human and animals and transmit a wide range of pathogens. The species identification of ticks is normally based on morphological keys. However, considering the importance of proper identification of the species and their bio-ecological characteristics, relying on morphological characteristics alone can be questionable. In this study, ITS2 and COI were selected for phylogenetic evaluation of Iranian species belonging to three genera: Dermacentor, Hyalomma and Rhipicephalus. Results: The ticks were from 10 populations from three different climatic and zoogeographical zones of Iran. The study of important morphological characteristics revealed notable differences in these characteristics between different populations of D. marginatus species.Contrary to the results of ITS2 sequence analysis which giving additional support for the view that D. niveus and D. marginatus should be viewed as one species only, the sequence analysis of COI and phylogeny favored the separation of the two species of D. niveus and D. marginatus. Given the greater importance of COI in identifying and distinguishing species, the Being two species should be considered.