Abilities to detect heterogeneity of ospC genotypes of the Lyme disease spirochete Borrelia burgdorferi in the tick vector by in vitro culture (IVC) and direct PCR (dPCR) were compared. IVC failed to detect one-third of the ospC genotypes detected by dPCR. Among IVC results, common ospC genotypes were overrepresented while occurrence of rare genotypes was underestimated.