B. burgdorferi invasiveness correlates with ospC genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospC heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3' ospC sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospC were seen in only one locality, and five of six sites contained two or more B. burgdorferi ospC clones. Four invasive patient isolates and six small mammal isolates clustered with "noninvasive" reference ospC genotypes. A high degree of ospC diversity exists among B. burgdorferi isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospC types contradicts the ospC invasiveness hypothesis. Alternative genetic markers for B. burgdorferi disseminated disease should be investigated.