A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.