Objective: To describe the characteristics of pulmonary tuberculosis patients with acid-fast bacilli (AFB) negative sputum and determine the value of Gene Xpert MTB/RIF of bronchial lavage fluid in diagnosis of sputum AFB-negative pulmonary tuberculosis. Method: This cross-sectional study was conducted on 120 patients who were admitted to the Respiratory Department of Thong Nhat Hospital from July 2022 to August 2023 suspected of pulmonary tuberculosis with AFB-negative sputum. Results: There were 67.9% male and 32.1% female. Mean age was 57.8 ± 20.1 years and the body mass index was 21.4 ± 1.8 kg/m2. Common comorbidities included hypertension (41.1%), type 2 diabetes (30.4%), and chronic lung disease (5.4%). There were 48.2% of patients who had injuries in more than 1 location on X-ray. Characteristics of X-ray injuries were in right lung (46.4%), left lung (28.6%), upper lobe (51.8%), lower lobe (17.8%). Infiltrative injury on X-ray accounted for 64.3%, followed by consolidation (21.4%) pleural effusion (10.7%), cavity (8.9%), and nodular (5.4%). Gene Xpert MTB/RIF of bronchial lavage fluid had tuberculosis bacteria in 40.8% of cases. The positive rate of Mycobacteria growth indicator tube culture (MGIT) of bronchial lavage fluid was 46.7%. The sensitivity of Gene Xpert MTB/RIF compared to MGIT was 85.7% and the specificity was 98.4%. The positive predictive value was 97.9% and the negative predictive value was 88.7%. Rifampicin resistance rate was 4.1%. Conclusion: Pulmonary tuberculosis patients with AFB-negative sputum have diverse clinical and radiological characteristics. Gene Xpert MTB/RIF of bronchial lavage fluid had high sensitivity and specificity in diagnosis of AFB-negative pulmonary tuberculosis. Rifampicin resistance rate was at a low rate. DOI: 10.59715/pntjmp.3.2.14