Abstract: Mycoplasma ovipneumoniae is associated with atypical pneumonia in sheep and goats. Traditional detection of M. ovipneumoniae can be complex owing to its slow growth and fastidious nutritional requirements. In present study, researchers developed a PCR Assay Method for specific and sensitive detection of M. ovipneumoniae using primers targeting the heat shock protein 70 gene (hsp70). The results demonstrated that the hsp70 gene-based PCR is specific by producing an amplicon of 135 bp only with M. ovipneumoniae strains. The detection limit is 4.4 pg which is 10 times more sensitive than the existing 16S rDNA-based PCR. The hsp70 gene-based PCR also demonstrated a higher detection rates for both nasal swabs and lung tissues when comparing with 16S rDNA-based PCR.