BACKGROUND A number of different viral, bacterial, fungal and protozoan organisms can cause atypical pneumonia; the three most common are Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae. Atypical pneumonias are by far the most underdiagnosed and underreported clinical entities, and very few studies have been reported in India. This study was conducted from March 2015 to September 2015 to determine the seroprevalence of M. pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies in patients with lower respiratory tract infections, and to compare IgM ELISA with that of the Sanger sequencing. Sequencing proved to be one of the rapid, sensitive and specific method of diagnosis in these cases. MATERIALS AND METHODS This cross sectional hospital based study was done at Government General and Chest Hospital, Erragadda, Hyderabad. The study population included 90 patients attending outpatient department and admitted as inpatients between March 2015 to September 2015 with a high probability of atypical pneumonia based on clinical symptoms and signs like fever, cough, dyspnoea, headache. Crepitations and presence of new pulmonary infiltrates on chest x-ray, in cases like COPD with acute exacerbation, bronchial asthma with acute exacerbation and Community-acquired pneumonia cases. Sputum and serum samples collected from each patient. 90 blood samples collected were subjected to ELISA to detect IgM antibody (EUROIMMUN) for Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae. The sputum samples showing no pathogenic organisms (NPO) were subjected to PCR for amplification of V3 region of 16 SMA followed by Sanger sequencing. RESULTS 17 cases were positive for IgM antibodies for atypical bacteria and 8 cases were positive by Sanger sequencing. The common presenting symptoms were cough, fever, chest pain, dyspnoea and headache. More number of cases were detected in Community-acquired pneumonia patients followed by COPD and bronchial asthma with acute exacerbation. CONCLUSION IgM ELISA having correlated clinically with reasonable sensitivity could be a useful tool in diagnosing atypical pneumonia caused by L. pneumophila, M. pneumoniae and C pneumoniae. Sanger sequencing in comparisons of low sensitivity although highly specific can be recommended for application in discriminating species and not for diagnosis from sputum samples.