Infections with feline hemotropic mycoplasmas (hemoplasmas) have been documented in domestic cats and free-ranging feline species with high prevalences in Iberian lynxes (Lynx pardinus), Eurasian lynxes (Lynx lynx), European wildcats (Felis silvestris silvestris), African lions (Panthera leo) in Tanzania and domestic cats in South Africa. The prevalence of hemoplasmas has not yet been investigated in free-ranging felids in southern Africa. In this study we screened 73 blood samples from 61 cheetahs in central Namibia for the presence of hemoplasmas using quantitative real-time PCR. One of the cheetahs tested PCR-positive. Phylogenetic analysis based on partial sequencing of the 16S rRNA and RNAse P genes revealed that the isolate belongs to the Mycoplasma haemofelis/haemocanis group. This is the first molecular evidence of a hemoplasma infection in a free-ranging cheetah.