In this study, we aimed to assess the in vitrosusceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitrosusceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosisisolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitrosusceptibility.