Cytauxzoon felis is a tick-borne protozoan parasite of considerable importance to domestic cats in the United States. Despite high mortality among clinically presenting cats, some cats may either recover from infection, becoming permanent chronic carriers of C. felis, or may not develop disease following exposure. C. felis in healthy (asymptomatic) cats is currently detected by qPCR or through observation of piroplasms in blood smears. The present study aims to compare the prevalence of C. felis in healthy populations of pet and free-ranging cats by using two different diagnostic tests: an indirect ELISA and a probe-based qPCR. Samples, along with demographic information, were collected collaboratively from healthy cats in the states of Oklahoma, Arkansas, and Missouri (USA) in either pet or free-range populations. Significantly more cats were detected to be C. felis positive by the ELISA than qPCR. Of the total of cats tested by ELISA and qPCR, 248 (35.37%) tested ELISA positive but qPCR negative, indicating formation of specific C. felis antibodies in cats with undetectable circulating C. felis DNA. The results of this study provide insights into the occurrence of C. felis in healthy cats that are exposed in the south-central United States and present a useful diagnostic strategy for detecting C. felis.