We measured the frequency of appearance of spontaneous mutants resistant to gentamicin, kanamycin, streptomycin, and spectinomycin in saprophytic and pathogenic Leptospira strains. The mutations responsible for the spontaneous resistance to streptomycin and spectinomycin were identified in the rpsL and rrs genes, respectively. We also generated a gentamicin resistance cassette that allows the use of a third selectable marker in leptospires. These results may facilitate further advances in gene transfer systems in Leptospira spp.