The performance of Western blots (immunoblots) prepared with eight strains of Borrelia burgdorferi representing B. burgdorferi sensu stricto, B. garinii, and B. afzelii genospecies was tested with a panel of sera with various clinical presentations collected from eight geographic regions. European sera were generally more reactive to blots prepared with B. garinii or B. afzelii strain antigens, in particular B. garinii 20047 and B. afzelii VS461. North American sera were more reactive with B. burgdorferi sensu stricto strains. Our observation of significant differences in the levels of reactivity of some sera on Western blots of certain strains is potentially important for the development and implementation of generic interpretive criteria. Preferential reactivity of sera from patients with nerve and/or palsy symptoms to B. garinii strains and with cutaneous disease to B. afzelii strains was observed. On the basis of our results, we have concluded that strain 20047 is the best strain to use for the development of a generic Lyme borreliosis Western blot for Europe.