Mycoplasma infection is a major problem in veterinary medicine and in poultry production. The pathogen has many strains, so that diagnosis of the disease using culture method is not effective. The objective of this work was to evaluate the prevalence of Mycoplasma gallisepticum (MG) in Kuwait poultry farms using serology and molecular methods in comparison to the culture under specific conditions. A total of 50 swab samples from choanal cleft and tracheal samples and blood samples were obtained from three different local farms, the blood samples were processed for an Enzyme Linked Immunosorbent Assay (ELISA) detection and the swab samples for Polymerase Chain Reaction (PCR) and culture methods detection. A PCR diagnostic kit (VenoMGs) and ELISA diagnostic kit (ProFLOK), were used in comparison to the traditional culture method, to study the spread of this disease in samples from broiler and layer flocks. Fifty chicken samples were tested for mycoplasmosis, samples tested with ELISA gave 24 positive (48%) and 29 were positive by PCR (58%) and only seven (14%) were positive with culture methods. Swab samples obtained from the choanal cleft gave more positive (60%) with PCR than tracheal samples (56.6%). The culture gave 20 and 5% positive, respectively for tracheal and choanal samples. The methods reported here are of high sensitivity and specificity for Mycoplasma. Both the PCR and ELISA methods are superior to culture method for detection of avian mycoplasmosis. This study showed that MG infection is prevalent in commercial broiler and layer chickens in Kuwait poultry farms. The use of these methods for surveillance of the disease will establish data concerning the predominant Mycoplasmosis diseases in Kuwait if done on a large scale.