This study aimed to determine the prevalence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in breeder flocks showing respiratory symptoms. A total of 77 flocks (2153 tracheal swabs and blood samples) were sampled and all were tested by MG real time PCR (MG-rPCR) and MG-ELISA, and 32 flocks were tested by MS real time PCR (MS-rPCR). In the first part of this study covering 28 flocks, all samples from chickens with marked clinical symptoms and high MG-antibody levels gave negative results with MG-rPCR(1). Therefore, the MG-lipoprotein gene-specific primers (MG-rPCR(1)) of this PCR were replaced with MG-16S rRNA primers (MG-rPCR(2)), as were the MS-16S rRNA primers (MS-rPCR), thus the study was pursued accordingly. All of the first 28 flocks, which were 100% positive by MG-ELISA, were MG-rPCR(1) negative, whereas in the second part of the study, other 49 flocks, which were 87.8% MG seropositive, were found 42.9% positive by MG-rPCR(2). In addition, 5 selected flocks from the first 28 were negative, whereas 7.4% of the 27 selected flocks from the second 49 were positive by MS-rPCR. Overall, 81 out of 432 MG-rPCR(1-2) (18.7%) performed from 77 flocks, and 13 out of 187 MS-rPCRs (6.9%) in 32 flocks were determined as positive. ELISA results indicated that there could be significantly high false-positives in serological tests, thus results should not be relied upon one test system. Also, this study revealed that, for the confirmation of Mycoplasma-infected flocks in laboratories, rPCR is a reliable method as long as suitable primers are selected, and that MG and MS prevalence is considerably high in winter season.