Mycoplasma pneumoniae( M.pneumoniae) is an important respiratory pathogen in humans. Few studies revealed that it had contributed to joints inflammation, and considered as a primary cause of acute polyarthritis. The presence of the organism in the synovial fluid of the patients with arthritis raises questions about the pathogenic role that this organism plays in disease development. Objective: the aim of this work is to investigate the presence of M.pneumoniae in the synovial fluid of non rheumatoid arthritis patients. Subject and Method: synovial fluid samples were aspirated from forty patients presenting with joint effusion. Each sample was subjected to cultivation on specific media, and polymerase chain reaction (PCR) using specific primers directed to P1 adhesion gene of M. pneumoniae. Results: M. pneumoniae was recovered by culture in 5/43 ( 11.6%) of synovial fluid samples ; 4/21 (19%) of osteoarthritis(OA) patients’ samples, 1/6 (16.7%) of unclassified arthritis patients’ samples . Using PCR, M. pneumoniae was detected in 7/43 (16.3%) of the samples) ; 6/21 (28.5% of OA patients’ samples), 1/6 (16.7%) of unclassified arthritis patients’ samples). Conclusion: M. pneumoniae may play a role in the development of arthritis and it could be responsible for progression into chronicity and exacerbation of arthritis. This study demonstrated that PCR is more sensitive and rapid compared to culture for detection of M. pneumoniae in the synovial fluid samples.