BACKGROUND AND ABJECTIVE Hemotropic Mycoplasma species are vector-borne bacteria that attach and grow on the surface of erythrocytes in various mammals, yet reports of canine and feline hemoplasmosis in Iran are scarce. The aim of this study was molecular detection and identification of hemoplasmas in the blood of dogs and cats from different regions of Iran. MATERIALS AND METHODS From December 2018 to February 2021, a total of 370 dogs from Hamedan, Kermanshah, Yazd, Amol and Ahvaz, and 361 cats from Tehran, Hamedan, Kermanshah, Yazd, Kerman and Mashhad were examined clinically, their bodies were searched for collection of ectoparasites, and cephalic or saphenous blood specimens were collected. Genomic DNA was extracted from blood and ectoparasite specimens and the presence of hemotropic Mycoplasma DNA was detected using group-specific, and identified using species-specific conventional PCRs detecting Mycoplasma haemocanis (Mhc), Candidatus Mycoplasma haematoparvum (CMhp), Candidatus Mycoplasma haemominutum (CMhm), Mycoplasma haemofelis (Mhf) and Candidatus Mycoplasma turicensis (CMt) followed by Sanger sequencing. Correlation of infection and risk factors (geographical area, keeping condition, body condition, sex, age, infestation with ectoparasite) were analyzed. RESULTS AND DISCUSSION In group-specific PCR, 210 dogs (56.7%) and 57 cats (15.7%) tested PCR-positive for hemotropic Mycoplasma. Species-specific PCR and sequencing revealed infection with Mhc in 17.8%, with CMhp in 7.02% and with both parasites in 31.9% of dogs. In cats, positivity rates of 10.2% for CMhm, 2.2% for Mhf, 0.9% for CMt and 2.50% for both CMhm and Mhf (co-infection) were recorded. Flea infestation, poor body condition, and being older than 3-years-old correlated with hemoplasmosis. Ectoparasites collected from dogs included Ctenocephalides canis and Pulex irritans fleas, Rhipicephalus sanguineus sensu lato ticks, Heterodoxus spiniger lice and Hippobosca longipennis keds in which DNA of hem
