Objectives: To assess the characteristics of M. tuberculosis strains based on the microbiology tests from the new pulmonary tuberculosis and re-treatment pulmonary tuberculosis patients, who have been assigned by first-line anti tuberculosis drugs. Methods: Perspective descriptive study was conducted on 64 new cases and 39 re-treatment pulmonary tuberculosis patients assigned for first line anti-tuberculosis drugs at Hanoi Lung Hospital and National K74 Hospital. The microbiology test results of sputum smears, culture by MGIT-BACTEC 960, GenXpertMTB/RIF and Drug Susceptibility Test were compared for the new-case and re-treatment pulmonary tuberculosis patient groups. Results: There was no significant difference of sputum smear results between re-treatment and new-case groups. The GU and TTD in MGIT-BACTEC results when culturing M. tuberculosis from sputum were widely variable between patients in both the groups. The re-treatment group had a higher tendency in TTD. Although all patients had the results as GenXpertMTB+/RIF- (sensitivity to Rifampicin), the drug susceptibility test Lowenstein-Jansen revealed that there were some patients with multi-drug resistance to first-line anti-tuberculosis drugs in both the new-case and re-treatment TB patient groups (1.56 and 12.82%, respectively). The rate of drug resistance to any first-line anti-tuberculosis drugs of Mycobacterium tuberculosis strains isolated from re-treatment tuberculosis patients is higher than that from new cases (53.85 and 21.88%, respectively). Conclusions: The Mycobacterium tuberculosis strains isolated from the re-treatment pulmonary tuberculosis patients had the higher rate of drug resistance to first-line anti-tuberculosis drugs than that of the new cases.