Lyme disease is the most commonly reported vector-borne disease in North America and Europe, yet we know little about which components of the causative agent, Borrelia burgdorferi, are critical for infection or virulence. Molecular genetics has provided a powerful means by which to address these topics in other bacterial pathogens. Certain features of B. burgdorferi have hampered the development of an effective system of genetic analysis, but basic tools are now available and their application has begun to provide information about the identities and roles of key bacterial components in both the tick vector and the mammalian host. Increased genetic analysis of B. burgdorferi should advance our understanding of the infectious cycle and the pathogenesis of Lyme disease.