Background: Tuberculosis (TB) is a curable infectious disease yet the leading cause of death worldwide and in Bangladesh it is responsible for 7% of total deaths every year.Objectives: This cross sectional study was conducted to isolate and identify Mycobacterium tuberculosis from Sputum and to evaluate the efficacy of polymerase chain reaction (PCR) for the early diagnosis of pulmonary tuberculosis.Patients and Methods: A total of 150 clinically suspected pulmonary TB patients (male/female: 97.53; age: 31.9±9.8 years) were enrolled in this study. Freshly passed single morning Sputum was collected from each patient and diagnostic efficacy of PCR was compared with staining and culture methods.Results: Among 150 Sputum samples, 25 (16.7%) were positive by Ziehl-Neelsen (ZN) stain, 37 (24.7%) yielded growth in Lowenstein-Jensen (LJ) media and 45 (30%) were positive by PCR. Of the 37 isolated Mycobacteria in culture, 36 were M. tuberculosis and one was identified as non-tuberculous Mycobacterium by PCR and biochemical tests. The mean detection time was 26.9 days for the culture and 12 hours for the PCR method. Considering the culture method as the gold standard, the sensitivity of PCR was 97.3% and specificity was 92% with accuracy of 93.3%. Twenty-one (16.8%) smear negative cases and nine (7.9%) culture negative samples were positive by the PCR method.Conclusions: PCR was the most sensitive, accurate and rapid method for diagnosis of TB. It was concluded that, for the diagnosis of pulmonary tuberculosis, PCR directly from Sputum may be recommended in Bangladesh, especially in suspected tuberculosis patients who remain negative by conventional methods.