In this study, the authors examined the technical performance of culture methodology using specific media: Mycoplasma isolation media of pleuropneumonia-like organisms (PPLO) broth and PPLO agar. Digitonin sensitivity, growth inhibition, the serum plate agglutination test, a commercially available enzyme-linked immunosorbent assay (ELISA) and a commercially available simplex polymerase chain reaction (PCR) test were used to detect Mycoplasma gallisepticum infections in samples collected from the lungs, trachea and tracheal swabs of poultry. These samples were collected from broiler-breeder flocks, broiler flocks and layer flocks. In addition, genomic bacterial deoxyribonucleic acid (DNA) was extracted and amplified, using a simplex PCR. The seroprevalence of M. gallisepticum antibodies in chickens and chicks was also investigated. The prevalence of M. gallisepticum was found to be highest in the layer flocks, at 33.3% (17/51), when the tracheal swab procedure was adopted. In young birds, the serum plate agglutination test and ELISA assay detected antibodies against M. gallisepticum in 69.9% (320/458) and 58.3% (267/458) of the chicken samples, respectively, and 48.7% (146/300) and 60% (180/300) of the samples from the chicks.