The experimental evolution (EE) of microbes has allowed evolutionary biologists to examine adaptive processes in real time, generating novel insights into fundamental laws of evolution. Less appreciated is the potential of this approach to advance our understanding of microbial cells and molecular processes as a complement to traditional molecular genetics. The tracking of mutations underlying phenotypic changes offers the opportunity for detailed molecular analyses of novel phenotypes. This provides a breadth of information on diverse biological systems and may retrace key past events of natural history. Here, we highlight how the field has advanced our understanding of gene regulation, antibiotic resistance, and host–microbiome interactions to exemplify how EE can be used to provide new light on microbial systems.