The aim of the study was bacteriological isolation of Mycoplasma gallisepticum (MG) and detecting specific DNA by the Polymerase Chain Reaction (PCR) through testing 96 tracheal swab samples of chickens. The serum plate agglutination test (SPA) was performed to detect antibodies to MG in the blood sera of the examined chickens. Specific DNA was detected in 47 (49%) of the 96 tracheal swabs by means of the M. gallisepticum DNA Test Kit (Flock Check, IDEXX), MG was isolated from 3 (3.1%) of the 96 trachea samples and SPA revealed that 10 (10.4%) of the 96 sera were sero-positive and 10 (10.4%) suspicious. The results indicated that, despite its common use as a screening test in field conditions, the SPA test alone is not sufficient in diagnosing MG infections in chickens. The PCR technique, however, thanks to its speed and reliability in routine diagnosis seems to be an alternative method to the difficult and time-consuming techniques of culturing MG.