This study aimed to elucidate the epidemiological status of hemoplasma infection and investigate the interaction between Theileria orientalis and hemoplasmas in Japanese Black breeding cows raised in the Kyushu and Okinawa regions. Blood samples were collected from 400 cows from 80 different farms in eight prefectures (five samples per farm and 10 farms per prefecture). Mycoplasma wenyonii (Mw), “Candidatus Mycoplasma haemobos” (CMh), and T. orientalis were examined by polymerase chain reaction (PCR) assay using whole blood samples. PCR results showed that 91.5% (366/400) of cows were positive for bovine hemoplasma: 40.3% were infected with Mw only, 9.5% with CMh only, and 41.8% with both species. T. orientalis was detected in 36% (144/400) of cows. The infection rate of T. orientalis was higher in the grazing group (P<0.001) than in the housed group, while the rate of CMh infection was higher (P<0.05) in the housed group than in the grazing group, suggesting that not only the tick but also other arthropod vectors may contribute to hemoplasma transmission. Although the cows with hemoplasma dual infection showed higher (P<0.05) white blood cell counts compared with hemoplasma-negative cows, there was no difference in hematologic parameters related to the anemia between the hemoplasma-positive and -negative animals. This may indicate that Japanese Black cattle could have resistance to the anemia caused by infection with hemoplasma.