In this study, 746 questing Ixodes (I.) ricinus ticks from eastern Slovakia and 187 ticks from southern Poland were investigated for infection with Borrelia (B.) burgdorferi sensu lato and different outer surface protein A (OspA) types by an improved restriction fragment length polymorphism (RFLP) analysis of the ospA gene. The method enables differentiation of both single and multiple infections with B. burgdorferi s.s. (OspA type 1), B. afzelii (OspA type 2), B. garinii (OspA types 3-8), B. valaisiana (subgroups I and II), B. lusitaniae, B. bissettii, and the recently described genospecies A14S. Broad heterogeneity in B. burgdorferi s.l. was found including all species and subtypes except for B. lusitaniae, B. bissettii, and genospecies A14S. Regional prevalence of B. burgdorferi s.l. varied between 8% and 22.5%. The most frequent species were B. garinii (45.4%) and, notably, B. burgdorferi s.s. (31.3%). I. ricinus nymphs harbored almost exclusively B. burgdorferi s.s. and B. garinii OspA type 4, while in adults a broad variety of B. burgdorferi s.l. types was found. Mixed infections were significantly more often in nymphs than in adult ticks. In all mixed infected nymphs, B. burgdorferi s.s. with OspA type 4 was present. These data strongly suggest that B. burgdorferi s.s. and B. garinii OspA type 4 are maintained in these areas by specific transmission cycles involving a so far undetermined vertebrate host which is frequently fed on by I. ricinus larvae. This improved method provides a reliable tool for epidemiological studies on the heterogeneity of B. burgdorferi species and OspA types, an important prerequisite for improved local risk assessment and for test- and vaccine development for Europe.