Background Linezolid (LZD) is an important antibiotic for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), but mutations related to linezolid resistance remain inadequately characterized. In this study we investigated the resistance-related mutations of clinical isolates from MDR and XDR TB patients who failed linezolid treatment, using wholegenome sequencing. Methods We used the Sensititre™ plate to determine the phenotypic drug susceptibility and minimum inhibitory concentration (MIC) of first- and second-line anti-TB drugs, while using resazurin microtiter assay (REMA) for the MIC of linezolid. A total of 25 sequential isolates, previously collected from four MDR/XDR patients who failed LZD treatment, were used in this study. All isolates had whole genome sequencing performed with the Illumina® platform. Results All four TB patients had a form of multidrug-resistant TB, which is defined as a form of TB resistant to at least rifampicin and isoniazid, the first-line anti-TB drugs. Each patients’ isolates collected within three months of beginning LZD treatment were susceptible to LZD, but the isolates from the corresponding patient collected after three months had higher MIC for LZD and developed mutations in LZD resistance linked genes (Table 1). Whole genome sequencing of all strains collected from each patient revealed mutations in the LZD resistance linked genes rplC, rrl, and Rv0678 (Table 2). Conclusions After a given period of time, all four patients acquired resistance to LZD. However, the LZD MIC was below 32ug/ml, a concentration observed in a previous study. The rplC gene affects the ribosomal protein L3, the rrl gene affects the 23S rRNA, and Rv0678 is the regulator for the MmpS5-MmpL5 efflux pump. The combination of mutations in these genes caused the high-level linezolid resistance in each serial clinical isolate.