The hard tick Hyalomma marginatum Koch, 1844 is the main vector of Crimean Congo hemorrhagic fever (CCHF). Although this disease is a serious public health concern in Turkey, there is still no information about population structure and genetic diversity of this tick species. In this study, the genetic structure of H. marginatum populations collected from nine different locations of Turkey were evaluated using five microsatellite markers. A total of 75 H. marginatum samples were collected from domestic animals and genotyped. We hypothesized that localities which have high CCHF incidence rates (Bayburt, Amasya and Tokat) might be genetically distinct from the localities with low incidence rates. All approaches used in this study (DAPC, STRUCTURE and F-ST) showed low levels of genetic diversity. Although, no genetic pattern was observed between localities that can be associated with CCHF incidence rates, there was moderate genetic differentiation between Igdir and Tekirdag populations (F-ST = 0.142) and also between Igdir and Mugla populations (F-ST = 0.128). DAPC confirmed this finding, as Igdir, Mugla and Tekirdag populations were found to be genetically different when compared to other populations. The mechanisms underlying overall observed low genetic variation and only moderate differentiation between some of these populations should be investigated in further studies in the context of other host-parasite-vector system regulating factors such as antropogenic transports of livestock, environmental conditions and the role of migratory routes of birds.