The prevalence and risk factors of 2 bovine hemoplasma species—Mycoplasma wenyonii (Mw) and “Candidatus Mycoplasma haemobos” (CMh)—were examined by direct PCR using whole blood samples collected from 343 cattle in Eastern Hokkaido, Japan. The sensitivity of the direct PCR assay was 10-fold higher than the standard PCR assay, detecting 5 copies of bovine hemoplasma DNA per reaction. An epidemiological survey of sampling location, living conditions, age and bovine leukemia virus antibody was performed to assess infection risk factors. The prevalence rates were 38.5% for Mw and 39.1% for CMh, respectively, with an overall prevalence rate of 64.7% for all bovine hemoplasma infections. Significant differences were found in sampling location, living conditions and age. Compared with pastured cattle, farmed cattle were significantly more susceptible to infection with total hemoplasma species (P<0.001), and were particularly susceptible to CMh (P<0.001). Cattle that were 1–3 years of age were more susceptible to infection with hemoplasma compared with other age groups. Bovine leukemia virus infection status was not significantly associated with hemoplasma infections. Blood examination revealed significantly lower RBC, Hb, and PCV levels, and a higher MCV in infected cattle than in noninfected cattle.