Background. Despite low sensitivity in detection of Mycobacterium tuberculosis, sputum acid-fast smear remains the main diagnostic method. This study aimed to compare the diagnostic performance of Xpert MTB/RIF assay versus conventional sputum acid-fast smear. Materials and Methods. A cross-sectional study was conducted at Chiang Mai University Hospital, Thailand. Patients who were ≥15 years old and had clinically suspected pulmonary tuberculosis were included. Results. 109 specimens from 57 patients were included. Using MGIT sputum culture as a reference standard, the sensitivity (SEN) and specificity (SPEC) for Xpert were 95.3% (95% CI, 84.2%, 99.4%) and 86.4% (95% CI, 75.7%, 93.6%). The SEN and SPEC for sputum acid-fast smear were 60.5% (95% CI, 44.4%, 75.0%) and 98.5% (95% CI, 91.8%, 100%). Xpert had significantly higher sensitivity (p value < 0.001) and lower specificity (p value = 0.022) than sputum acid-fast smear. Among 43 culture-proven M. tuberculosis specimens, sensitivity of Xpert was 100% (95% CI, 86.7%, 100%) in acid-fast positive smears (n = 26) and 88.2% (95% CI, 63.5%, 98.5%) in acid-fast negative smears (n = 17). Conclusions. The good sensitivity and specificity of Xpert assay in detecting M. tuberculosis from sputum specimens may help in early diagnosis and treatment of pulmonary tuberculosis, particularly among patients who had acid-fast negative sputum smear.