Mycoplasma spp. are hemoparasites with potential to cause anemia and other debilitating conditions in domestic cats. The aim of the study was to detect by molecular techniques hemoplasma infection in cats from metropolitan area of Rio de Janeiro, Brazil, and establish its correlation to hematological data. From a total of 197 cats sampled, 22.8% were positive by real-time PCR and 11.2% by blood smear. The infection frequency of hemoplasma species determined by PCR using species-specific primers was 4.6%, 4.6% and 11.7% for Mycoplasma haemofelis (Mhf), 'Candidatus M. turicensis' (CMt) and 'Candidatus Mycoplasma haemominutum' (CMhm), respectively. Therefore, CMhm was the species with the highest incidence. Coinfection between Mhf and CMhm (1%), Mhf and CMt (0.5%) and CMt and CMhm (0.5%) was observed, as well as triple coinfection in one animal (0.5%). Regarding hematological changes associated with M. haemofelis infection, it was found macrocytic hypochromic anemia (p < 0.01), lymphocytosis (p < 0.03), thrombocytopenia (p < 0.04) and the presence of activated monocytes (p < 0.04). On the other hand, no hematologic parameter relevant to feline mycoplasma infection was associated with CMhm and CMt infection. Male cats were more prone to be infected by Mhf and CMhm (p < 0.01), as well as adult cats (p > 0.05). The results reveal the occurrence of three species of haemotropic mycoplasmas at metropolitan area of Rio de Janeiro and denote Mhf as an important cause of anemia in cats. Consequently, the knowledge about the disease and the factors related to its occurrence are required for the adoption of preventive action aiming at animal and man health.