MLST appears best in population genetic study but it is expensive. Due to the sequence conservation in housekeeping genes, MLST sometimes lacks the discriminatory power to differentiate bacterial strains, which limits its use in epidemiological investigations. To improve the discriminatory power of MLST, a multi-virulence-locus sequence typing (MVLST) approach has been developed using Listeria monocytogenes . MVLST broadens the benefits of MLST but targets virulence genes, which may be more polymorphic than housekeeping genes. Population genetics is not the only relevant factor in an epidemic. Virulence factors are also important in causing disease, and population genetic studies struggle to monitor these. This is because the genes involved are often highly recombining and mobile between strains in comparison with the population genetic framework. Thus, for example in Escherichia coli, identifying strains carrying toxin genes is more important than having a population genetics-based evaluation of prevalent strains.
The advent of second-generation sequencing technologies has made it possible to obtain sequence information across the entire bacterial genome at relatively modest cost and effort, and MLST can now be assigned from whole-genome sequence information, rather than sequencing each locus separately as was the practice when MLST was first developed.  Whole-genome sequencing provides richer information for differentiating bacterial strains (MLST uses approximately 0.1% of the genomic sequence to assign type while disregarding the rest of the bacterial genome).  For example, whole-genome sequencing of numerous isolates has revealed the single MLST lineage ST258 of Klebsiella pneumoniae comprises two distinct genetic clades, providing additional information about the evolution and spread of these multi-drug resistant organisms, and disproving the previous hypothesis of a single clonal origin for ST258.