Background Infections with the three feline haemotropic mycoplasmas Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis cause feline infectious anemia. The purpose of this study was to investigate the prevalence of carriage of feline haemoplasma in Danish cats in different age groups. The presence was detected by a conventional polymerase chain reaction (PCR) assay on blood samples as well as by real-time PCR (RT-PCR). Results The study revealed a prevalence of 14.9% Candidatus Mycoplasma haemominutum positive cats and 1.5% Mycoplasma haemofelis positive cats. No cats were found positive for Candidatus Mycoplasma turicensis. The results showed a statistically significant higher prevalence in older (>8 years) cats compared to younger cats and a higher prevalence among domestic cats compared to purebred cats. As part of this study, we developed a cloning strategy to obtain Danish positive controls of haemoplasma 16S rRNA. Conclusion From convenience-sampled cats in Denmark, we found that 16.4% were carriers of feline haemotropic mycoplasmas. Haemoplasma was mostly found in older and domestic cats. The prevalence found in Denmark is similar to that found in several other European countries.