Eight ixodid tick species, associated with 59 free-ranging mammals belonging to 10 species, were collected at five different localities in the Free State Province, South Africa. Four of the study areas were nature reserves (Willem Pretorius, Sandveld, Tussen-die-Riviere, and Soetdoring), and one site was a private farm located in Senekal district. The collection was performed from March 2006 until June 2006. Ticks (n=569) and tissues from animals (n=52) were analyzed by polymerase chain reaction, reverse line blot, and sequencing for various tick-borne pathogens belonging to the genera Babesia, Theileria, Anaplasma, and Ehrlichia. Rhipicephalus (Boophilus) microplus, the known vector of Babesia bovis responsible for Asiatic redwater in South Africa, was found for the first time in the Free State Province. Rhipicephalus appendiculatus also was collected in areas in the Free State where it has not been previously described. Anaplasma marginale was detected for the first time in a gemsbok (Oryx gazella gazella). Gene sequences recovered in this study were 98–100% homologous with GenBank sequences for Anaplasma bovis, Theileria separata, and Theileria sp. Malelane sable antelope.