An epidemiological survey of Iriomote cats (Prionailurus bengalensis iriomotensis) was conducted to understand the prevalence and molecular characteristics of hemotropic mycoplasma (hemoplasma). A series of ecological surveys of Iriomote cats were performed between November 2003 and September 2010. During this period, 31 Iriomote cats were captured or found, and 39 blood samples were collected. Polymerase chain reaction screening for hemoplasmas and BLAST searches revealed that 4 of the 31 cats were positive for hemoplasma infection (n=3, Mycoplasma haemofelis [Mhf]; n=1, 'Candidatus M. turicensis' [CMt]). The 4 infected cats were captured or found in the northern area of the island of Iriomote. Phylogenetic analyses revealed close relationships between Mhf and CMt isolated from Iriomote cats compared with those from domestic cats and other wild felids. In our study, we identified two species of hemoplasma in Iriomote cats. The number and location of the hemoplasma-positive cats appeared to be limited; however, continuous surveillance of hemoplasma infection in Iriomote cats is necessary.