Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres. The overall organization of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes. We present evidence here that a rather recent nonhomologous recombination event in the B. burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which is present in the strain B31 lineage. At least two successive rounds of addition of linear plasmid genetic material to the chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism by which plasmid genetic material can become part of the chromosome. The unusual nonhomologous nature of this rearrangement suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B. burgdorferi chromosomes.