BackgroundThree feline hemoplasma species are recognized: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’. These species can cause anemia in cats and have a worldwide distribution.ObjectivesThere was no previous information on hemotropic mycoplasma spp in cats in Iran and the Middle East. Accordingly, we investigated the molecular presence, and clinical signs and hematological profile in cats infected with these microorganisms in Iranian cats.MethodsPolymerase chain reaction (PCR) assays and cytology were performed on 100 blood samples collected from Iranian Shorthair cats. ACBC and case history were also collected for each sample.ResultsBy PCR, 22 (22%; 14–30%, 95% CI) samples were positive. The prevalence of M haemofelis, ‘Ca M haemominutum’, and ‘Ca M turicensis’ was 63.63% (14/22), 54.54% (12/22), and 18.18% (4/22), respectively. Some double and triple co‐infections were also found. Using PCR as the reference method, cytology had poor sensitivity (27%) and reasonable specificity (89.74%). Male cats were at a higher risk of infection (P = .001). Cats older than 8 years were more frequently infected than the younger cats (P = .0018). Lower HCT (P = .018), RBC count (P = .028) and HGB concentration (P = .003) were also associated with hemoplasma PCR‐positive status.ConclusionsBased on this study, the most prevalent feline hemoplasma species in Iranian cats was M haemofelis, but double and triple co‐infections are also documented. Age and sex, as well as reduced RBC parameters, were predisposing factors for hemoplasma infection.