Fifty-three southern USA Borrelia isolates were characterized using randomly amplified polymorphic DNA fingerprinting analysis (RAPD). Twenty-nine types were recognized among 37 B. andersonii strains, seven types among eight B. bissettii strains, and seven types among seven B. burgdorferi sensu stricto strains. Strain TXW-1 formed a separate RAPD type. Nearly complete sequences of the rrs genes from 17 representative southern Borrelia were determined. The similarity values were found to be 96-100% within the B. burgdorferi sensu lato (s.l.) complex, 94-99% among the relapsing fever borreliae, and 93-99% between the two complexes. Phylogenetic analysis indicated that all the Borrelia strains we analyzed could be divided into two parts: the B. burgdorferi s.l. complex and the relapsing fever borreliae complex. TXW-1 segregated with the North American relapsing fever borreliae and formed a separate subbranch.