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Mycoplasma gallisepticum (MG) infections cause significant economic losses to the poultry industry worldwide. Vaccination with live and inactivated vaccines and administration of antibiotics are commonly used as control measures. Herein, we describe a 27-wk-old broiler-breeder vaccinated with live TS-11 MG vaccine at 10 wk of age. The flock subsequently exhibited mild respiratory signs and a slight decrease in egg production at 27 wk of age. Serological examination of serum samples using a serum plate agglutination test revealed 32% positive samples 6 wk after vaccination and 100% positive samples after the onset of clinical disorders. Detection of MG by polymerase chain reaction and partial sequencing of the second cytadhesin-like protein encoding gene (mgc2) showed 99% nucleotide sequence identity with MG field strains and a lower identity with the TS-11 vaccine strain. We conclude that vaccination of chickens with TS-11 against MG will not prevent infection with field strains. The serum plate agglutination test could be used to indicate field infection in flocks vaccinated with TS-11. However, the molecular detection and differentiation of MG strains are essential in controlling MG infection in poultry.