Mycoplasma bovis is a highly contagious major mastitis pathogen with multiple clinical presentations in dairy cows. This kind of mastitis does not respond to available antibiotics and actually there is no effective therapy for this infection, thus the best way of prevention and control is to diagnose and cull the affected cows in the herd. The objective of this study was to detect Mycoplasma bovis in bulk tank milk samples by nested PCR in Mashhad,Iran. One hundred and four fresh bulk tank milk samples from 52 dairy herds were collected four weeks apart. Mycoplasma bovis was not detected from any of them by either direct PCR on milk or after enrichment in modified Hayflick’s broth. Two other mycoplasma species were detected after enrichment and one other mycoplasma species without enrichment by mycoplasma spp. primer. Sequencing of the PCR products from two positive samples confirmed the presence of mycoplasma that were Mycoplasma canadense and Mycoplasma yeatsii.

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