Rocky Mountain spotted fever (RMSF) caused by Rickettsia rickettsii is a severe rickettsiosis that occurs in nearly every state of the continental USA. RMSF is endemic in Central and Southern America, with recent well-documented cases in Mexico, Costa Rica, Panama, Colombia, Brazil, and Argentina. RMSF is the most malignant among known rickettsioses causing severe multiorgan dysfunction and high case fatality rates, which can reach 73% in untreated cases. Variations in pathogenic biotypes of R. rickettsii isolates have been described, and potential correlations of these differences to various clinical manifestations of RMSF have been suggested. We have recently reported on a method of genetic comparison employing sequence differences in intergenic regions (IGR typing) in isolates of R. rickettsii of human, tick, and animal origin. The grouping obtained correlated well with 2 other genotyping systems we have developed, which target the presence and distribution of variable numbers of tandem repeats (TR) and insertion/deletion (INDEL) events. Twenty-five total genotypes of R. rickettsii in 4 primary groups could be distinguished: isolates from Montana, isolates associated with Rhipicephalus sanguineus ticks and human infections in Arizona, other isolates from the USA where Dermacentor variabilis is thought to be the primary vector, and the isolates primarily associated with Amblyomma ticks from Central and South America. In addition, isolate Hlp#2, which is often considered to be a nonpathogenic isolate of R. rickettsii and closely related serotype 364D, exhibited the most diversity from the other isolates compared, and they differ significantly from each other. Because complex interactions underlie the pathogenesis of R. rickettsii in vivo, it is difficult to define the causality of individual events that occur in infected vertebrate hosts and humans. Many microbial factors are likely to contribute to the varied ability of R. rickettsii to cause cellular injury; some of them may also contribute importantly to its virulence for vertebrate hosts and may be linked to the variable genetic markers we have identified. Since circulation of R. rickettsii in nature includes vertical transstadial and transovarial transmission within tick vectors and horizontal passages through vertebrate hosts, it is plausible that isolates of different virulence arose when they became isolated during adaptation to novel vertebrate and tick hosts. Characterization of the physiologically important changes in rickettsial gene expression that occur immediately after tick-to-human or tick-to-animal transitions may require development of new experimental systems.