Early diagnosis of Borrelia burgdorferi infection, hampered by the absence of detectable antibodies in most patients with erythema chronicum migrans is important to prevent late-stage neurologic, rheumatologic, and skin disorders. Furthermore, B. burgdorferi has been claimed to be the causative agent in localized scleroderma (morphea). We used PCR amplification to search for B. burgdorferi outer surface protein OspA-specific sequences in DNA obtained from lesional skin biopsies on Finnish patients with clinically suspect erythema chronicum migrans, lymphocytoma, morphea, or with diverse skin manifestations and persistent high antibodies to B. burgdorferi flagellar antigen. Seronegative patients with other skin lesions served as controls. The amplicons obtained with primers specific for B. burgdorferi type strain B31 ospA sequence did not hybridize to the corresponding probes, and thus the DNA amplified from a Finnish B. burgdorferi erythema chronicum migrans skin isolate was sequenced. This 98-nucleotide sequence of ospA (332-429) showed 11% to 14% nucleotide divergence compared with the North American type strain (B31), several European strains, and an East Siberian tick strain. The sequence was almost identical (99%) to a Swedish isolate from acrodermatitis chronica atrophicans. Using oligonucleotides specific for the Finnish strain, a positive polymerase chain reaction-based hybridization was obtained in six of seven untreated erythema chronicum migrans patients infected in Finland or in Estonia, and in the lymphocytoma patient. Only two of the erythema chronicum migrans patients had IgG or IgM antibodies to flagellin. However, all seven morphea lesions as well as the other lesions were polymerase chain reaction negative. Polymerase chain reaction-based hybridization of B. burgdorferi OspA gene from skin-derived DNA thus provides a sensitive and specific diagnostic tool. In conditions not unequivocally known to be caused by B. burgdorferi, like in morphea, this assay was negative. We also demonstrate that peri-Baltic B. burgdorferi isolates show homology in their OspA genes but differ from geographically more distant isolates.