Due to prevailing environmental and climatic conditions South Africa hosts one cheetah subspecies (Acinonyx jubatus jubatus) and a wide range of tick-borne protozoa such as Babesia. Blood samples collected from 143 cheetahs at four study sites, namely the Ann van Dyk Cheetah Breeding Center-De Wildt (Brits and Shingwedzi), the Cheetah Outreach and the Hoedspruit Endangered Species Centre, were examined for Babesia infection. The V4 hypervariable region of 18S rRNA gene was amplified and subjected to the Reverse Line Blot (RLB) hybridisation assay. Hybridisation of the parasite DNA with Babesia genus and species-specific probes was evident. The results showed a predominance of Babesia lengau (n=63, 44.1%), followed by Babesia felis (n=3, 2.1%) and Babesia canis rossi (n=7, 4.8%). Unfed ixodid ticks (n=10,432), collected from the vegetation by drag-sampling, represented five species: Amblyomma hebraeum, Amblyomma marmoreum, Haemaphysalis elliptica, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus simus and Rhipicephalus zambeziensis,. The monthly occurrence of ixodid ticks at the De Wildt Cheetah Breeding Centre (Brits) showed a higher activity in the warm months of the year. Recovery of ticks decreased during the warm hours of the day, suggesting that free-living ticks are humid dependent. The presence of birds, rodents, free-ranging antelopes such as nyalas (Tragelaphus angasii), kudus (Tragelaphus strepsiceros), bushbucks (Tragelaphus scriptus) and impalas (Aepyceros melampus), as well as Burchell’s zebras (Equus burchellii) and leopard tortoises (Geochelone pardalis) can contribute to the availability of various tick species at the breeding centres. Mice as the host for immature instars of ixodid tick species and unfed ixodid ticks were studied for presence of Babesia species. Babesia lengau was detected in 22 (39.2%) mice as well as in Haemaphysalis elliptica larvae, nymphs and adults. The presence of B. lengau in mice suggests a long-term association since the host preference of B. lengau for mice remains unclear. However, the presence of this parasite in unfed imature and adult H. elliptica is indicative of a transstadial transmission suggesting that this tick species may be a potential vector for B. lengau. The correlation between Babesia infection and various parameters such as gender, age, tick burdens and location, in two different breeding farms belonging to the De Wildt Cheetah Breeding Centre was analysed using the Fisher’s exact test analysis. The prevalence of Babesia species in cheetahs was associated with tick burden suggesting a strong positive correlation between the prevalence of infection and presence of suspected vector ticks. Regardless of tick burden, age could be related to prevalence of infection, meaning that the fact that older cheetahs had a higher prevalence of infection with Babesia species. These findings were of considerable interest especially since at the time of study the cheetahs in both populations did not show clinical signs of infection with Babesia species.