Pathogenic Mycoplasmas have been found in humans, animals, plants and insects,which can cause many diseases. However, the diagnosis of Mycoplasmas with culture and serological methods is limited, due to slow growth, extensive nutritional requirements and cross antigens of the pathogens. In recent years, with the progress of molecular biology, polymerase chain reaction (PCR) has been adapted widely for the diagnosis of Mycoplasma infections. This review mainly summarizes the successful application of PCR techniques, such as standard PCR, PCR ELISA, nested PCR, real time quantitative PCR, RT PCR and multiplex PCR to the diagnosis of Mycoplasmas . These techniques provide timesaving, sensitive, specialized methods in Mycoplasmas detection.