A single set of oligonucleotide primers was designed from known 16S ribosomal RNA (rRNA) sequences of Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. iowae (MI). This set of primers selectively amplifies a 780-base-pair DNA fragment within the 16S rRNA gene of MG, MS, and MI but does not amplify other avian mycoplasmas or other bacteria. The detection limit of the multi-species polymerase chain reaction (PCR) was approximately 100 mycoplasma (MG, MS, MI) colony-forming units per PCR reaction. The PCR product was differentiated by restriction fragment length polymorphism with the restriction enzymes HpaI, HpaII, and MboI. Preliminary results from field samples suggest that this technique could be a useful and rapid diagnostic test for the detection of these three pathogenic poultry mycoplasmas.