BACKGROUND: Tuberculosis, caused by bacteria in the Mycobacterium tuberculosis complex (MTBC), is a major global public health burden. Strain-specific genomic diversity in the known lineages of MTBC is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Fast and accurate tracking of MTBC strains is therefore crucial for infection control, and our previous work developed a 62-single nucleotide polymorphism (SNP) barcode to inform on the phylogenetic identity of 7 human lineages and 64 sub-lineages. METHODS: To update this barcode, we analysed whole genome sequencing data from 35,298 MTBC isolates (~ 1 million SNPs) covering 9 main lineages and 3 similar animal-related species (M. tuberculosis var. bovis, M. tuberculosis var. caprae and M. tuberculosis var. orygis). The data was partitioned into training (N = 17,903, 50.7%) and test (N = 17,395, 49.3%) sets and were analysed using an integrated phylogenetic tree and population differentiation (FST) statistical approach. RESULTS: By constructing a phylogenetic tree on the training MTBC isolates, we characterised 90 lineages or sub-lineages or species, of which 30 are new, and identified 421 robust barcoding mutations, of which a minimal set of 90 was selected that included 20 markers from the 62-SNP barcode. The barcoding SNPs (90 and 421) discriminated perfectly the 86 MTBC isolate (sub-)lineages in the test set and could accurately reconstruct the clades across the combined 35k samples. CONCLUSIONS: The validated 90 SNPs can be used for the rapid diagnosis and tracking of MTBC strains to assist public health surveillance and control. To facilitate this, the SNP markers have now been incorporated into the TB-Profiler informatics platform ( https://github.com/jodyphelan/TBProfiler ).