Our planet is an increasingly urbanized landscape, with over half of the human population residing in cities. Despite advances in urban ecology, we do not adequately understand how urbanization affects the evolution of organisms, nor how this evolution may affect ecosystems and human health. Here, we review evidence for the effects of urbanization on the evolution of microbes, plants, and animals that inhabit cities. Urbanization affects adaptive and nonadaptive evolutionary processes that shape the genetic diversity within and between populations. Rapid adaptation has facilitated the success of some native species in urban areas, but it has also allowed human pests and disease to spread more rapidly. The nascent field of urban evolution brings together efforts to understand evolution in response to environmental change while developing new hypotheses concerning adaptation to urban infrastructure and human socioeconomic activity. The next generation of research on urban evolution will provide critical insight into the importance of evolution for sustainable interactions between humans and our city environments.