BACKGROUND: Feline hemotropic mycoplasmas are parasites of erythrocytes and include three species, Mycoplasma haemofelis, Candidatus mycoplasma haemominutum and Candidatus mycoplasma turicensis. Diagnosis of the infection with these microorganisms can be carried out using conventional assays such as blood cytology. However, these assays have a low accuracy and a high rate of false-positive results due to the poor techniques and procedures and high occurrence of artifacts. Therefore, molecular techniques such as polymerase chain reaction (PCR) are better methods for the diagnosis of infections by these bacteria. OBJECTIVES: The purpose of the present study was to evaluate Feline hemotropic mycoplasma prevalence and phylogenetic analysis in Tehran. METHODS: Sixty cat blood samples were collected from veterinary clinics in Tehran from 2011 to 2012. Giemsa stained blood smears have been examined by the light microscopes and the positive samples were used for DNA extraction and PCR. Positive PCR samples were sequenced for the differentiation of bacterial species and phylogenetic analysis. RESULTS: Thirty-two samples were positive in direct examination from which two samples were identified as M. haemofelis by the PCR. No positive samples of C. M. haemominutum or C. M. turicensis were found in PCR. Phylogenetic analysis of the isolates showed that these isolates were more similar to the isolates from China and Thailand compared to those from other countries. CONCLUSIONS: This study is the first report of phylogenetic analysis of hemotropic mycoplasmas in Iran. Based on the high sequence similarity between Iran, China and Thailand isolates, it can be concluded that these bacteria possibly had the same origin.