Hemotropic mycoplasmas are a group of bacteria of the Mycoplasma genus that do not have a cell wall and cannot be cultured. Three species are known to infect cats; Mycoplasma haemofelis, ´Candidatus Mycoplasma haemominutum and ´Candidatus Mycoplasma turicensis. The mode of transmission of these bacteria is not fully understood; it is assumed that the main role in transmission is through vectors and contact with blood that occurs during aggressive interactions between cats. After infection, these bacteria are attach to host's erythrocytes and can cause immunemediated hemolytic anemia. The prevalence of feline hemotropic mycoplasma infection varies significantly in certain geographical areas, and there is a complete lack of data on the prevalence in Croatia. Therefore, the aim of this research was to apply the molecular method of polymerase chain reaction and determine the frequency of infection with hemotropic mycoplasmas in cats - patients of the University Veterinary Hospital. In addition, possible risk factors for infection were investigated. A total of 105 archived whole blood samples of cats, collected during the spring and summer of 2021, were analyzed for the presence of hemotropic mycoplasmas using conventional polymerase chain reaction (PCR). An overall prevalence of 22.86% was determined. The frequency of infections was significantly higher in anemic animals, and a higher probability of infection was found in male cats and in older animals in general. Animals infected with feline immunodeficiency virus were also statistically significantly more susceptible to hemotropic mycoplasma infection. Our results are the beginning of research on the molecular detection of hemotropic mycoplasmas in cats in Croatia. For further research, more detailed, structured anamnestic data should be used, it is necessary to analyze a larger number of risk factors for infection and to determine the clinical significance of infection with certain types of hemotrophic mycoplasmas.