There is a need for a better understanding of the epidemiology of Mycoplasma synoviae (MS) infection in broiler breeders in Brazil. Many features of the infection remain unrecognizable, because there are no clinical signs of the disease. A detailed testing was performed at each 6 to 8 weeks in three MS-free flocks introduced in farms with endemic MS infection for a follow-up epidemiological study. Every flock was monitored by polymerase chain reaction (PCR), by serum plate agglutination (SPA) and hemagglutination inhibition (HI) for serology studies, and isolation of mycoplasmas from tracheal swabs. PCR was found to be the most sensitive test, detecting early MS infection. Serology was positive in less than 50% of the sera and MS was isolated only between 27 and 28 weeks of age and in a maximum of 60% positive hens. A similar profile was seen for MS infection in all three flocks. Infection started at brooding, whereas laboratory detection of the assymptomatic infection was more probable in the weeks of increasing egg production. This predictable profile during rearing may be very useful for the optimization of monitoring MS infection in broiler breeder flocks.