Understanding the local tick species composition is crucial for overcoming the diseases they transmit. A comprehensive survey integrating molecular identification was conducted in the eastern and southeastern parts of Turkey, where tick surveys have previously been neglected. A total of 596 specimens belonging to four tick genera were collected from 27 localities in Turkey during the summers of 2019 and 2020. Seventy-seven representative individuals were chosen for molecular analysis. Nine distinct species, Rhipicephalus bursa, Rhipicephalus turanicus, Rhipicephalus rossicus, Hyalomma asiaticum, Hyalomma excavatum, Hyalomma marginatum, Hyalomma aegyptium, Haemaphysalis sulcata, and Dermacentor marginatus were identified. The presence of R. rossicus was demonstrated for the first time in Turkey. Two lineages of R. turanicus were identified, and representatives of both lineages were recorded. Our Hyalomma phylogenetic tree was consistent with previous findings from Turkey; however, new sympatric areas for Hy. marginatum and Hy. excavatum and Hy. marginatum and Hy. asiaticum were recorded. Two haplotypes (Haemaphysalis sp. and Dermacentor sp.) could not be identified using morphological and molecular methods. In addition to making a valuable contribution to the molecular database of ticks in the Middle East, this study will also stimulate comparative studies on the genetic structure, ecology, and vector competence of different populations of these species in Turkey as well as in other parts of the world.