We developed a high-throughput method based on terminal restriction fragment length polymorphisms (T-RFLP) to identify ospC genotypes from field-collected samples of Borrelia burgdorferi. We first validated the method by analyzing B. burgdorferi ospC previously identified by sequencing. We then analyzed and compared ospC genotypes detected from ear biopsy tissue from natural populations of the white-footed mouse, a major B. burgdorferi reservoir host species in the eastern United States, and larval ticks feeding on those individual mice. The T-RFLP method enabled us to distinguish all 17 ospC genotypes tested, as well as mixed samples containing more than one genotype. Analysis costs compare favorably to those of alternative ospC identification methods. The T-RFLP method will facilitate large-scale field studies to advance our understanding of genotype-specific transmission patterns.