The spirochete Borrelia burgdorferi is a tick-borne pathogen that causes Lyme disease. Although B. burgdorferi sensu lato is a diverse group of bacteria, only three genospecies, B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are known to be pathogenic and commonly recognized to cause human disease. To assess the potential of another common genospecies, Borrelia bissettii, to induce disease, a mouse model was employed. Two Colorado isolates of B. bissettii (CO-Bb) induced lesions of the bladder, heart, and femorotibial joint 8 weeks after inoculation into mice. In contrast, two British Columbia (BC-Bb) isolates, could not be cultured or amplified by PCR from target organs, and did not induce lesions. Consistent with pathology and culture results, the antibody response in mice to BC-Bb was minimal compared to CO-Bb, indicating either transient localized infection or rapid immune clearance of BC-Bb. Although sequence analysis of the rrf (5S)-rrl (23S) intergenic spacer region indicated 99% homology between CO-Bb and BC-Bb, polyacrylamide gel electrophoresis (PAGE) analysis indicated five distinct protein differences between these low-passage isolates. These studies support the prospect that B. bissettii may indeed be the causative agent of Lyme borreliosis cases in Eastern Europe, associated with the atypical Borrelia strain 25015, and in other regions. To our knowledge, this is the first evidence that B. bissettii can induce pathology in a vertebrate host.