Background: Although infections of cats with H. felis, H. silvestris and H. canis have been described, there was no information on the prevalence of feline hepatozoonosis in Iran. The aim of this study was the molecular detection and identification of Hepatozoon in the blood of cats from six provinces. Methods: From 2018 to 2022, blood samples were collected from 772 cats (299 stray, 473 client-owned) cats from both sexes and different ages in Tehran (n = 295), Mashhad (n = 239), Kermanshah (n = 85), Hamedan (n = 53), Yazd (n = 52) and Kerman (n = 48). The presence of Hepatozoon was detected using conventional PCR. Positive samples were further examined with Sanger sequencing and phylogenetic analysis. Statistical analysis was performed to identify risk factors associated with infection. Results: 29 cats (21 stray, 8 owned) scored PCR-positive. Infected cats were found in Mashhad (n = 20; prevalence = 8.4%), Hamedan (n = 2; prevalence = 3.8%), Kermanshah (n = 2; prevalence = 2.4%) and Tehran (n = 5; prevalence = 1.7%). BLAST analysis of obtained nucleotide sequences revealed H. felis (n = 25) and H. canis (n = 3). Hepatozoonosis was significantly higher in stray cats, and in Mashhad. There was no statistically significant association between the infection and age or sex. Conclusion: This research is the largest epidemiological study on feline hepatozoonosis in Iran. Further investigations on domestic and wild felids in other regions of the country, on the clinical impact of the infection, and possible presence of H. silvestris are suggested.
