Amblyomma americanum collected near Lake Wappapello, Missouri, tested positive for Borrelia lonestari using polymerase chain reaction and sequence analyses of B. lonestari 16S rRNA and flagellin (flaB) genes. Twelve pools containing a total of 214 nymph or adult ticks contained evidence of infection with B. lonestari (minimum prevalence 5.6%). These data suggest that persons in southeast Missouri are at risk for exposure to B. lonestari after A. americanum tick bite, a possible cause of erythema migrans-like rash illness in this region. Derivation of the complete coding sequence for B. lonestari flaB is also reported.