Hemoplasma infections are emerging and wild fauna can represent an important reservoir of these pathogens. However, there are very few epidemiological studies about the occurrence of hemoplasmas in wild cats around the world. The purpose of this study is twofold: (1) evaluate the occurrence and phylogeny of hemoplasmas in captive wild felines at a zoo in the state of Paraná, Brazil, and (2) verify the correlation between subpopulations of these bacteria and the hematological and biochemical parameters of the animals. PCR was used to detect hemoplasmas in the blood of three cougars (Puma concolor), a jaguar (Panthera onca), a tiger (Panthera tigris) and a lion (Panthera leo), followed by sequencing and phylogenetic analysis. The cougars and jaguar were found to be hemoplasma-positive by PCR. The phylogenetic analysis of the 16S rRNA gene sequences enabled the identification of genotypes of ‘Candidatus Mycoplasma haemominutum’ circulating in this zoo. The identified sequences were closely related to hemoplasma sequences originating from domestic cats and other wild cats, but the infected cougars and jaguar were healthy and showed no hematological or biochemical changes. It was concluded that P. concolor and P. onca are exposed to ‘Candidatus Mycoplasma haemominutum’ in Paraná, but further research is suggested to assess the resistance of wild cats to different hemoplasma subpopulations.