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Objectives: Tick-borne diseases have emerged as an increasing medical problem in the world. Being the most prevalent ixodid ticks in Europe, Ixodes ricinus and Dermacentor reticulatus are responsible for transmission of numerous zoonotic pathogens (e.g., human granulocytic anaplasmosis and Lyme borreliosis). Despite their public health significance, studies on the prevalence of tick-borne agents are scare for Eastern Europe. The objective of this study was to examine the prevalence of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Borrelia burgdorferi sensu lato (B. burgdorferi s. l.) in ixodid ticks from Southeastern Ukraine. Methods: Over a 5-year period (2014–2018), 358 questing and 389 engorged ixodid ticks were collected from Southeastern Ukraine (Zaporizhzhya region). The ticks were identified as Dermacentor marginatus, D. reticulatus, I. ricinus, and Rhipicephalus rossicus. Nucleic acid samples extracted from tick pools were subjected to RT-PCR analyses for A. phagocytophilum, E. chaffeensis, and B. burgdorferi s. l. Results: The examined ixodid ticks tested negative for the aforementioned pathogens with the exception of I. ricinus ticks. For questing I. ricinus ticks, minimum infection rates of A. phagocytophilum and B. burgdorferi s. l. were, respectively, 4.2–7.7% and 8.6–12.7%. Conclusions: These findings will be valuable for medical and veterinary practitioners when risks associated with tick-borne diseases are assessed for southeastern regions of Ukraine. [ABSTRACT FROM AUTHOR]