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Certain Mycoplasma spp. are pathogens of poultry, but little is known of the role of mycoplasmas in disease of birds of prey. Species-specific polymerase chain reactions (PCRs) for the detection of the poultry pathogens Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae were therefore evaluated for use in birds of prey. The specificities of the PCR methods were established using avian and other mycoplasmas and also selected walled bacteria. The sensitivities of the different PCR assays varied between 100 fg and 10 pg DNA. Fifty-three tracheal swabs from healthy captive and free-ranging birds of prey were then investigated using these PCRs, and in no case was an amplicon obtained for M. gallisepticum/M. imitans, M. iowae or M. synoviae. Species-specific primers for M. meleagridis amplified a product from eight birds of prey but restriction enzyme analysis as well as sequencing of PCR products demonstrated these results to be false positives. Alignment studies of the sequenced products with the 16S rRNA gene sequence of various Mycoplasma species in GenBank demonstrated an identity of 91% to M. meleagridis but of 98% to Mycoplasma buteonis or Mycoplasma gallopavonis. Isolation and attempted identification of these mycoplasmas suggested it may be a previously unrecognized species.