Hemoplasma is a tribal name for epierythrocytic mycoplasmas including Mycoplasma suis and M. parvum which are currently recognized in pigs as causative of porcine hemoplasmosis. Here, we report a real-time PCR assay for differential detection of these swine hemoplasma species by using allelic primers in the16S rRNA gene, and its application to survey for hemoplasma infections in pigs. Universal primers and species-specific primers were designed and evaluated by using swine blood samples positive in hemoplasmas. Mycoplasma suis and M. parvum infections were both confirmed by universal primers, and mixed infections were clearly distinguished by species-specific primers. Further, we applied this real-time PCR assay to 120 swine blood specimens from clinically healthy pigs in eleven farms in Japan, and found six (5.0%) were positive for M. suis and 18 (15.0%) were positive for M. parvum, and three (2.5%) were mixed infection by both hemoplasma species.