The transformation of DNA sequencing technologies has enabled more powerful and comprehensive genetic profiling of microbes. The sheer number of informative loci provided by genome-sequencing allows the investigation of structural variation and horizontal gene transfer as well as delivering novel insights into genetic origins, evolution and epidemiological history. Microbial genomes can be sequenced en masse at high coverage but have associated challenges of high mutation rates and low conservation of genome structure. Consequently, detecting changes in DNA sequences requires a nuanced approach specific to the organism, availability of similar genomes, and types of variation. Here, we outline the high power of genome-sequencing to detect a wide scope of polymorphism classes. Samples without related species on which to scaffold a genome sequence require specific assembly methods that can be enhanced by progressive procedures for improvement. Polymorphism identification depends on genome structure, and error rates in closely related specimens can be reduced by incorporating population-level information. The development of genome analysis platforms is hastening the optimization of variant discovery and has direct applications for pathogen surveillance. Robust variant screening facilitates more sensitive scrutiny of population history, including the origin and emergence of infectious agents, and a deeper understanding of the selective processes that shape microbial phenotypes.