Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic infection of biomedical significance, caused by Borrelia burgdorferi sensu lato (s.l.) spirochetes and transmitted by Ixodes species ticks. It usually circulates among wildlife vertebrate reservoirs and vector ticks but may infect humans, causing multisystem problems. In far western and northern North America, the host reservoirs, tick vectors, and genospecies of Borrelia are well known but not so in the southern U.S., where there is controversy as to the presence of "true" LD. Here we report the presence of the LD spirochete B. burgdorferi sensu stricto (s.s.) and Borrelia bissettii, three main reservoir hosts, and two enzootic tick vectors in the southeastern U.S. The two enzootic tick vectors, Ixodes affinis and Ixodes minor, rarely bite humans but are more important than the human biting "bridge" vector, Ixodes scapularis, in maintaining the enzootic spirochete cycle in nature. We also report extraordinary longevities and infections in the reservoir rodents Peromyscus gossypinus, Sigmodon hispidus, and Neotoma floridana.