In the past 10 years, microbiology has undergone a revolution that has been driven by access to cheap high-throughput DNA sequencing. It was not long ago that the cloning and sequencing of a target gene could take months or years, whereas now this entire process has been replaced by a 10 min Internet search of a public genome database. There has been no single innovation that has initiated this rapid technological change; in fact, the core chemistry of DNA sequencing is the same as it was 30 years ago. Instead, progress has been driven by large sequencing centers that have incrementally industrialized the Sanger sequencing method. A side effect of this industrialization is that large-scale sequencing has moved out of small research labs, and the vast majority of sequence data is now generated by large genome centers. Recently, there have been advances in technology that will enable high-throughput genome sequencing to be established in research labs using bench-top instrumentation. These new technologies are already being used to explore the vast microbial diversity in the natural environment and the untapped genetic variation that can occur in bacterial species. It is expected that these powerful new methods will open up new questions to genomic investigation and will also allow high-throughput sequencing to be more than just a discovery exercise but also a routine assay for hypothesis testing. While this review will concentrate on microorganisms, many of the important arguments about the need to measure and understand variation at the species, population and ecosystem level will hold true for many other biological systems.