Hemotrophic mycoplasmas (HM) are small, cell wall-less bacteria and infections are known for a wide range of animals. One possible indication of equine HM infection was given in 1978, when a 'haemobartonellosis' outbreak was diagnosed in Nigerian horses by microscopy. However the first molecular proof of HM in horses was not reported until 2010, when a fragment of about 900bp of the 16S rRNA of the equine HM was obtained. This sequence was used for the development of a SYBR green I real-time PCR assay specific for equine HM. The lower detection limit of the PCR was ten 16S rDNA copy numbers per ml of blood. The newly designed assay was successfully applied for the detection and quantification of HM in horses in Germany. A high sample prevalence of 26.5% (95% CI: 18.8-35.5%) was found (31 out of 117 horses). The mean bacterial load was 1.10×10(6) 16S rDNA copy number/ml blood (range: minimum 1.05×10(3), maximum 1.27×10(7)). Equine HM were also detected by microscopy (Giemsa and acridine orange stained blood smears), but results do not correlate very well with PCR results, as microscopy proved rather unspecific and not sensitive. In horses younger than one year, a significant correlation between PCR positive status and anemia was found. No correlation was found in PCR-positive animals older than one year. Therefore we assume that HM infection has a higher clinical relevance in young animals.