Objectives: To explore the drug susceptibility of levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ) and delamanid (DLM) against multidrug resistant tuberculosis (MDR-TB) isolates from drug resistance survey of southwest China, and to illustrate the genetic characteristics of MDR-TB isolates with acquired drug resistance. Methods: A total of 339 strains were collected from smear-positive TB patients in the drug resistance survey of southwest China between January 2014 and December 2016. The MICs were determined for MDR-TB identified by conventional drug susceptibility testing. Genes related to drug resistance were amplified with their corresponding pairs of primers. Results: MDR was observed in 88 (26.0%; 88/339) isolates. LFX had the highest resistance rate (50.0%; 44/88), followed by MFX (38.6%; 34/88). The resistance rate to LZD, CFZ, and DLM was 4.5% (4/88), 3.4% (3/88), and 4.5% (4/88), respectively, and the lowest resistance rate was observed in BDQ (2.3%; 2/88). Of the 45 isolates resistant to LFX and MFX, the most prevalent resistance mutation was found in gyrA with the substitution of codon 94 (34/45, 75.6%). All two CFZ - BDQ cross resistant strains had a mutation in the Rv0678 gene. Two of four LZD resistant isolates carried mutations in rplC gene. Of the four isolates resistant to DLM, one isolate with the mutations in codon 318 of fbiC gene and two isolates in codon 81 of ddn gene. Conclusion: This study provided evidence for further application of new anti-TB drugs in the treatment of MDR-TB in China.