Next-generation DNA sequencing technologies can generate unprecedented amounts of genomic data, even for non-model organisms. Here we describe how these new technologies have facilitated recent key advances in ecology and evolutionary biology, and highlight several outstanding ecological and evolutionary questions that are distinctly suited to the innovations they provide. Importantly, using these technologies to their full potential requires careful experimental design and critical consideration of several caveats associated with them. Although several significant challenges remain to be resolved before the integration of next-generation sequencing technologies into single-investigator research programs, we argue that they will soon transform ecology and evolution by fundamentally changing the ranges and types of questions that can be addressed.