Mycoplasama gallisepticum (MG) and Mycoplasma Synoviae (MS) are important avian pathogens which cause respiratory diseases resulting in large economic losses in Syria and world-wide poultry industry. The objective of this study is using multiplex polymerase chain reaction for direct detection of these agents from respiratory infections in broiler breeder flocks at Hama Province in Syria. For this purpose, 203 tracheal swabs were collected from birds which belonging to 29 flocks suffered from respiratory symptoms during the continued period between May 2011 and May 2012. The results revealed that 27 flocks (93%) were infected by these species (16 mixed infections and 11 single infections). The number of infected flocks with MG was 25 flocks and MG prevalence in these flocks was 86%. While the number of infected flocks with MS was 18 flocks and MS prevalence in these flocks was 72%. Regardless of the flocks which belonging to them the studied samples, the results showed that 128(63%) birds were infected with MG and MS (66 mixed infections and 64 single infections);119 birds were infected with MG , MG prevalence in  the studied birds was 59% and 75 birds were infected with MS, MS prevalence in the studied birds was 37%.