An immunoglobulin G immunoblot was developed with antigenic extracts of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana genospecies and was reacted with sera from patients with neuroborreliosis, acrodermatitis, and Lyme arthritis. A detailed analysis of the reactivities of the protein bands was performed, and a two-step scoring procedure was selected to determine the preferential reactivity of sera to one particular genospecies. The discriminative potential of 5 proteins (12-kDa, 16-kDa, 18-kDa, OspA, and 66-kDa proteins) was used as a rapid first-step scoring method, followed by scoring of 14 additional protein bands if necessary. The advantage of this procedure is the low percentage of serum samples with inconclusive results for one of the four species (10% for patients with neuroborreliosis, 6% for patients with acrodermatitis chronica atrophicans, and 6% for patients with Lyme arthritis). Among 31 serum samples from patients with neuroborreliosis, 16 were more reactive to B. garinii, 7 were more reactive to B. afzelii, 3 were more reactive to B. valaisiana, and 2 were more reactive to B. burgdorferi sensu stricto. Of 31 serum samples from patients with acrodermatitis, 26 showed a higher level of reactivity to B. afzelii. Of 34 serum samples from patients with Lyme arthritis, 21 were more reactive to B. burgdorferi sensu stricto, 10 were more reactive to B. afzelii, and 1 was more reactive to B. valaisiana. Our results suggest an organotropism of Borrelia species and provide some evidence of a pathogenic potential of B. valaisiana in humans.