Restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction-amplified gene fragments was used to characterize 24 isolates of spotted fever group rickettsiae previously identified as Rickettsia sibirica from their serologic properties. These strains were obtained in Russia between 1946 and 1991 from humans and different species of Ixodid ticks. The RFLP analysis was performed using amplified DNA products obtained with a genus-specific primer pair derived from the R. prowazekii citrate synthase gene and two group-specific primer pairs from the R. rickettsii 190-kD and 120-kD surface protein antigen genes followed by Alu I, Pst I, and Rsa I restriction endonuclease digestions. Although some differences were detected in biological characteristics among the examined strains, only a single R. sibirica genotype was found with these molecular tools of identification.