Since modernization and expansion of the poultry industry, infections with Mycoplasma spp. bacteria have been reported as a cause of considerable economic losses. The prevalence of Mycoplasma synoviae infection in 974,000 Portuguese broiler breeders, belonging to 36 flocks, was investigated from December 2008 to March 2012. This study was conducted using a commercial indirect enzyme-linked immunosorbent assay (ELISA) for the analysis of serum antibodies, and a polymerase chain reaction (PCR) for the tracheal tissue. Twenty-four flocks were simultaneously found positive by ELISA and PCR [66.7%, 95% confidence interval (CI): 43.5-76.9%]. The M. synoviae prevalence among chickens averaged 40.3% (483/1,200), with values ranging from 0.0 to 83.3% per flock. The prevalence of farms where M. synoviae positive birds have been found was determined in different poultry categories such as density, biosecurity, strains, offspring quality, premises'age, and others husbandry factors. Prevalence values were significantly higher among birds housed in new facilities (less than 3 years old) and were also significantly higher in the production period. The high prevalence of M. synoviae infection detected in the present study suggests the need to adopt appropriate control measures.