Ovine eperythrozoonosis is a zoonosis caused by Mycoplasma ovis(M.ovis) which parasites on erythrocytes and plasma.At present,there are several methods to diagnose eperythrozoonosis,but the result is not specialfor M.ovis and usually omits some infected carriers.The objective of this study was to establish a sensitive and specific method to detect M.ovis and investigate the epidemiology of this pathogen in Hubei Province of China.According to published GenBank 16S rRNA gene sequence (registration number EU165509.1)ofM.ovis,two pairs of primers were designed and the semi:nested PCR was developed.The final amplified fragment was 439bp and the sequencing result showed that it was 99% similar to the EU 165509.1.The assay exhibited high sensitivity approximately 10:12 fg and the specificity analysis revealed this assay had no cross reaction with M.suis,M.wenyonii,M.pneumonia of sheep,Theileria sergenti,as well as Babesia orientalis,particularly.A total of 371 blood samples,collected from seven different districts in Hubei Province of China,were examined for the presence of M.ovis using both conventional PCR and semi:nested PCR.Conventional PCR revealed that 97/371 samples were positive,whereas 151/371 samples were positive by semi:nested PCR assay.This result showed that the semi:nested PCR was much more sensitive than the conventional one.Statistical analysis found that the positive rate of the area and sex were not significantly different (p>0.05).The present study demonstrated that the semi:nested PCR is suitable for the early diagnosis of latent infected carriers and the investigation of epidemiology ofM.ovis.