The six reference strains of Mycoplasma iowae (I, J, K, N, Q and R) and 12 field strains, most of them isolated from turkeys, were studied with a growth-inhibition test and a dot immunobinding test with rabbit antisera to the different serovars of M iowae, 16S rDNA gene amplification by polymerase chain reaction, and pathogenicity for chicken or turkey embryos. Antigenic tests tended to be strain specific and showed that most field strains were closely related to serovars K or N. The two pairs of primers chosen in 16S rDNA guided the amplification of 332 base pairs (bp) or 892 bp fragments from all the M iowae strains tested. All the field strains tested were highly pathogenic for turkey embryos.