Bites from the hard tick Amblyomma americanum are associated with a Lyme disease-like illness in the southern United States. To identify possible etiologic agents for this disorder, A. americanum ticks were collected in Missouri, Texas, New Jersey, and New York and examined microscopically. Uncultivable spirochetes were present in ∼2% of the ticks. Borrelia genus-specific oligonucleotides for the flagellin and 16S rRNA genes were used for amplification of DNA. Products were obtained from ticks containing spirochetes by microscopy but not from spirochete-negative ticks. Sequences of partial genes from spirochetes in Texas and New Jersey ticks differed by only 2 of 641 nucleotides for flagellin and 2 of 1336 nucleotides for 16S rRNA. Phylogenetic analysis showed that the spirochete was a Borrelia species distinct from previously characterized members of this genus, including Borrelia burgdorjeri. Gene amplification could be used to detect these spirochetes in ticks and possible mammalian hosts.