Transposon mutagenesis is a powerful technique for generating random mutations. Previous studies had shown that the mariner transposon Himar1 could be utilized to introduce mutations in high-passage, noninfectious strains of Lyme disease Borrelia; however, transposon mutagenesis has not been reported in low-passage, infectious strains. In this pilot study, Himar1 transposon mutants were generated by transforming the infectious, highly transformable B. burgdorferi B31 clone 5A18 NP1 with the transposon vector pMarGent. The data provide 'proof in concept' that random transposon mutagenesis can be utilized for the global analysis of genes involved in the pathogenesis of Lyme disease Borrelia.