Antibiotic resistance is not a clinical problem with Lyme disease, but it has been extensively employed to genetically dissect the causative agent Borrelia burgdorferi. The first selectable marker was a coumermycin A1-resistant gyrB allele, which encodes a subunit of DNA gyrase, a target of several antibiotics. The utility of coumermycin A1 resistance has been compromised by technical and genetic barriers; resistance to other antibiotics has replaced the gyrB marker. Fluoroquinolones are another class of antibiotics that target DNA gyrase, as well as its homolog topoisomerase IV. Fluoroquinolone resistance in B. burgdorferi maps to parC, which encodes a subunit of topoisomerase IV, suggesting that this enzyme is the primary target of fluoroquinolones in Borrelia. A fluoroquinolone-resistant parC allele has been fashioned into a counter-selectable marker, a genetic tool used to select for the loss of DNA. One of the second-generation selectable markers is a heterologous aadA gene that confers resistance to spectinomycin and streptomycin, which target the small subunit of the ribosome. Selection with spectinomycin failed due to a high frequency of mutants in the population. These had mutations in 16S rRNA and were able to compete with wild type in vitro. This lack of a significant fitness cost for the mutant may contribute to the spread of antibiotic resistance.