Lyme disease in the United States is caused by the bacterial spirochete Borrelia burgdorferi s.s. (Johnson, Schmid, Hyde, Steigerwalt, and Brenner), which is transmitted by tick vectors Ixodes scapularis (Say) and I. pacificus (Cooley and Kohls). Borrelia lonestari, transmitted by the tick Amblyomma americanum L., may be associated with a related syndrome, southern tick-associated rash illness (STARI). Borrelia lonestari sequences, reported primarily in the southeastern states, have also been detected in ticks in northern states. It has been suggested that migratory birds may have a role in the spread of Lyme disease spirochetes. This study evaluated both migratory waterfowl and nonmigratory wild turkeys (Meleagris gallopavo silvestris, Eastern wild turkey) for B. burgdorferi and B. lonestari DNA sequences. A total of 389 avian blood samples (163 migratory birds representing six species, 125 wild turkeys harvested in habitats shared with migratory birds, 101 wild turkeys residing more distant from migratory flyways) were extracted, amplified, and probed to determine Borrelia presence and species identity. Ninety-one samples were positive for Borrelia spp. Among migratory birds and turkeys collected near migration routes, B. burgdorferi predominated. Among turkeys residing further away from flyways, detection of B. lonestari was more common. All A. americanum ticks collected from these areas were negative for Borrelia DNA; no I. scapularis were found. To our knowledge, this represents the first documentation of B. lonestari among any birds.