Genetic sequences characteristic of Borrelia lonestari (Barbour et al. 1996) were detected in two pools of adult Amblyomma americanum (L.) from Tennessee, corresponding to an estimated minimum field infection rate of 8.4 infected ticks/1000 adults. DNA amplification was conducted using primers derived from the B. lonestari flagellin gene that would also amplify Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner). Species-specific, internal probes were then used to differentiate between genetic sequences of the spirochetes. Subsequent nucleotide sequencing confirmed the presence of B. lonestari in A. americanum; B. burgdorferi was not detected. This represents the first report of B. lonestari from Tennessee, and suggests that Lyme-like illness may occur in Tennessee.