Microfluidic devices can mimic naturally occurring microenvironments and create microbial population heterogeneities ranging from planktonic cells to biofilm states. The exposure of such populations to spatially organized stress gradients can promote their adaptation into complex phenotypes, which are otherwise difficult to achieve with conventional experimental setups. Here a microfluidic chip that employs precise chemical gradients in consecutive microcompartments to perform microbial adaptive laboratory evolution (ALE), a key tool to study evolution in fundamental and applied contexts is described. In the chip developed here, microbial cells can be exposed to a defined profile of stressors such as antibiotics. By modulating this profile, stress adaptation in the chip through resistance or persistence can be specifically controlled. Importantly, chip‐based ALE leads to the discovery of previously unknown mutations in Escherichia coli that confer resistance to nalidixic acid. The microfluidic device presented here can enhance the occurrence of mutations employing defined micro‐environmental conditions to generate data to better understand the parameters that influence the mechanisms of antibiotic resistance.