Aim: To evaluate the performance of VITEK®MS with DNA sequencing for laboratory diagnosis of non-tuberculous mycobacteria (NTM) species in a resource-limited setting. Methods: 16SrRNA sequencing and MALDI-TOF mass spectrometry (VITEK®MS) was performed at a tertiary-care hospital in India. MALDI-TOF results were confirmed by 16S rRNA sequencing. In addition, sequencing of the internal transcribed spacer region was performed on slowly growing NTM. Results: Commonest species isolated were M. abscessus, M. intracellulare, M. avium, M. fortuitum and M. simiae. 16S rRNA sequencing and MALDI-TOF results had agreement of 94.5% for rapidly growing and 77.5% for slowly growing NTM. Conclusion: There is good correlation between VITEK®MS and sequencing for rapidly growing NTM. For slowly growing species, sequencing would be required in a third isolates.