This study was undertaken to compare the ability of four clinically used methods to detect the heteroresistance of the tubercle bacillus to Rifampicin (Rif). A total of 94 sputum smear-positive samples of re-treated tuberculosis (TB) patients were employed. Rif-resistant tubercle bacillus tests were carried out by four methods: Lowenstein-Jensen cultures (L-J), reverse transcription-polymerase chain reaction (RT-PCR), microscopic observation drug susceptibility (MODS), and high-resolution melting (HRM). The rates of detection of Rif-resistant bacteria by of L-J, RT-PCR, MODS, and HRM were 35.1% (33), 36.4% (35), 36.2% (34), and 16.0% (15), respectively. The rate of detection by RT-PCR was slightly higher than that of L-J and MODS (P > 0.05) and significantly higher than that of HRM (P < 0.01). The median detection time of L-J, RT-PCR, MODS, and HRM was 60 d, 1 d, 9 d, and 1 d, respectively. Smear-positive TB can be directly and rapidly detected using RT-PCR and MODS technologies. The positive rates of RT-PCR and MODS were not only highly consistent with that of L-J and remarkably higher than that of HRM, but also were obtained in a markedly shortened time. The combination of MODS and RT-PCR has a synergistic effect on the speed of detection of heteroresistance, which is of great value for the timely and accurate diagnosis, guiding the clinicians in formulating the optimal treatment of smear-positive pulmonary tuberculosis.