Mycoplasma synoviae (M. synoviae) infection is a cause of great economic loss in commercial egg layer hens. The aims of this study were to investigate the prevalence of M. synoviae and to compare the characteristics of M. synoviae infected and free flocks of commercial layers. A total of 400 tracheal swabs and 400 blood serum samples were collected from 20 different layer flocks. Random Amplified Polymorfic DNA (RAPD) analysis was used for the molecular typing of M. synoviae isolates to determine the source of infection. M. synoviae was isolated from 89 tracheal swabs collected from 5 out of 20 flocks. The genetic similarity between field strains ranged from 53% to 100% as determined by RAPD analysis. All M. synoviae strains separated into 2 main-clusters in UPGMA dendogram. These results revealed that the 5 infected flocks were contaminated from 2 different sources. The egg production of positive flocks was statistically lower (P<0.05) than that of the pathogen free flocks. Infection was more frequent in multi-age farms and on sites with several houses. The mortality of infected flocks was higher than uninfected flocks, but this difference was not statistically significant. The mean weight of eggs and the average live weight of hens were similar in free and infected flocks. In conclusion, M. synoviae infection significantly decreased egg production in layer hens and was more frequent in multiage farms.