Mycoplasma infections are of great concern in avian medicine, because they cause economic losses in commercial poultry production. A multiplex polymerase chain reaction (PCR) was optimized to simultaneously detect four pathogenic species of avian mycoplasmas. Four sets of oligonucleotide primers specific for Mycoplasma gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI) were used in the test. By using agarose gel electrophoreses for detection of the PCR-amplified DNA products, the sensitivity of detection was between 1 pg for MG, 1 pg for MS, 100 fg for MM and 100 pg for MI after 35 cycles of PCR. Similar sensitivity of these primers was achieved with broth cultures of these four organisms.