Background Over the last few decades tick-borne diseases have emerged as an increasingly significant public health problem. The distribution and prevalence of zoonotic pathogens infecting ixodid ticks from Western Europe have been extensively examined. However, data on ticks and tick-borne pathogens in Eastern Europe, particularly Ukraine are scarce. The objective of the current study was, therefore, to investigate the prevalence rates of Anaplasma phagocytophilum , the Anaplasmataceae family, Rickettsia spp., Babesia spp., Bartonella spp., and Borrelia burgdorferi sensu lato in engorged and questing ixodid ticks collected from five administrative oblasts (regions) of Ukraine, namely Chernivtsi, Khmelnytskyi, Kyiv, Ternopil, and Vinnytsia.Methods The ticks were collected from both wild and domestic animals and from vegetation. Of 524 ixodid ticks collected, 3, 99, and 422 ticks were identified as Ixodes hexagonus , Ixodes ricinus, and Dermacentor reticulatus , respectively. DNA samples individually extracted from 168 questing and 354 engorged adult ticks were subjected to pathogen-specific PCR analyses.Results The mean prevalence rates in all I. ricinus and D. reticulatus ticks were, respectively: 10.3% (10/97) and 2.8% (12/422) for A. phagocytophilum ; 69.1% (67/97) and 52.1% (220/422) for the Anaplasmataceae family; 24.7% (24/97) and 27.7% (117/422) for Rickettsia spp.; 3.1% (3/97) and 1.4% (6/422) for Babesia spp.; and 9.3% (9/97) and 4.7% (20/422) for Bartonella spp. Overall, between the five cities, there was no significant difference in the prevalence rates of any of the pathogens for the respective ticks ( p >0.05). The prevalence of B. burgdorferi s.l. in the engorged and questing I. ricinus ticks varied from 26.7% and 0% to 44.4% and 14.3%, respectively, with no statistical significance identified between the five cities ( p >0.05).Conclusions In addition to reporting the updated data for Kyiv and Ternopil, this study is the first to provide the prevalence rates of the tick-borne pathogens for Chernivtsi, Khmelnytskyi, and Vinnytsia. This investigation is also the first to detect Ca. N. mikurensis in ixodid ticks from Ukraine. These new data will be useful for medical and veterinary practitioners as well as public health officials when diagnosing infections and when implementing measures to combat tick-borne diseases in Ukraine.