Lyme disease, the most commonly reported arthropod-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. The acute stage of the infection has a varied presentation, ranging from mild, localized disease (characterized by a skin lesion) to highly symptomatic, disseminated disease. We hypothesize that this is due, in part, to B. burgdorferi genotypic variation. By combining PCR amplification of the 16S-23S rDNA spacer with restriction fragment length polymorphism (RFLP) analysis, several genotypes were identified among B. burgdorferi clinical isolates obtained from either skin or blood of early Lyme disease patients. Hematogenous dissemination in humans is associated with a distinct genotype and disease severity and spirochete burden was also associated with this same genotype in a murine model of Lyme disease. A genomic approach was undertaken to elucidate the differences in genome content and/or gene expression that may result in disease variability. Comparative transcriptional profiling of two clinical isolates with distinct genotypes (invasive and attenuated) was performed using whole genome arrays. A total of 78 ORFs had significantly different expression levels in the two isolates. Nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have considerably different cell surface properties. Comparative genome hybridization demonstrated that genotypic variation largely results from differences in plasmid content and/or sequence and revealed several plasmid-encoded candidate genes that are uniquely absent in attenuated strains. A number of genes identified in these investigations are currently under further study by genetic analysis to substantiate a possible role in virulence.