Mycoplasma contaminants can be considered important not only for their roles as pathogens but also they may indicate that insufficient care has been taken during vaccine manufacture or quality control. The purpose of this study was to test the poliomyelitis vaccines for Mycoplasma by culture and polymerase chain reaction (PCR) methods. During 2008 to 2009, a total of 47 lots of oral poliomyelitis vaccines were produced by Razi Vaccine and Serum Research Institute (RVSRI) in Iran. They were evaluated by culture and PCR for detection of Mycoplasma. In culture method, PPLO broth and PPLO agar medium were used and in PCR method, universal primers that were selected from the region 16S ribosomal RNA of Mycoplasmas were applied. In culture method, no changes on pH and color in PPLO broth tubes and no colony growth on PPLO agar plates were seen. In PCR method, Mycoplasma DNA was not detected in any of the tested vaccines. It was concluded that the current culture method for Mycoplasmas is reliable to detect viable Mycoplasmas in oral poliomyelitis vaccines, but our results confirmed the use of PCR assay as an efficient supplement to culture method.