Cytauxzoon felis is an intraerythrocytic apicomplexan of felids enzootic in the southeastern United States. In domestic cats (Felis catus), this parasite can result in the highly fatal disease cytauxzoonosis or bobcat fever. Bobcats (Lynx rufus) are the wild animal reservoir host. To date, the characterization of prevalence of C. felis in bobcats is mostly based on broad-scale surveys from hunter-harvested specimens collected across large geographic areas, usually consisting of multiple states. Detailed studies on the development, transmission, distribution, effects, and prevalence of C. felis in the tick vectors are scarce. To fill some of these gaps in the literature, such as prevalence in ticks and bobcats in a discrete region, we examined bobcats and ticks in an 8,000-km2 portion of southern Illinois. We screened for C. felis using a nested polymerase chain reaction that amplifies a fragment of the nuclear small subunit (SSU) 18S rRNA. We screened 125 individual bobcats collected in southern Illinois from 2003 to 2015; of these, 70.6% were positive for C. felis. In addition, we screened 214 ticks of both vector species (Amblyomma americanum and Dermacentor variabilis) and detected C. felis in 15.6% of them; this prevalence is higher than reported by previous surveys. Our study reports the prevalence of C. felis in ticks and bobcats from south Illinois. We found that 70.6% of bobcats and 15.6% of ticks were infected with C. felis, which suggests risk of transmission to domestic cats.