Mycoplasma hyorhinis (M. hyorhinis) is considered an etiological agent of arthritis in suckling pigs. Recently, some M. hyorhinis strains were shown to produce pneumonia that is indistinguishable from the mycoplasmosis caused by M. hyopneumoniae. In this study, we developed a sensitive and specific PCR assay to detect M. hyorhinis and applied the developed PCR assay for detection of Mycoplasma infection in clinical piglets infected with M. hyorhinis. We developed a new PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, designed from the Mycoplasma 16S-23S rRNA internal transcribed spacer (ITS) region. The primers and probe for the assay were designed from regions in the Mycoplasma 16S-23S rRNA ITS unique to M. hyorhinis. The developed PCR assay was very specific and sensitive for the detection of M. hyorhinis. The assay could detect the equivalent of 1 pg of target template DNA, which indicates that the assay was very sensitive. In addition, M. hyorhinis PCR assay detected only M. hyorhinis and not any other Mycoplasma or bacterial spp. of other genera. The new developed PCR assay effectively detected M. hyorhinis infection in pigs. We suggest that this PCR assay using a M. hyorhinis-specific primer pair, Mrhin-F and Mrhin-R, could be useful and effective for monitoring M. hyorhinis infection in pigs.