To determine whether the 23S rRNA mutation that confers macrolide resistance is present in >1 Treponema pallidum strain, 58 isolates collected between 2001 and 2005 were screened for this mutation and for an unrelated sequence that distinguishes between strains. The odds of identifying a macrolide-resistant strain increased over time (P=.006). In subjects who had received macrolides in the previous year, the relative risk of harboring a resistant strain was 2.2 (95% confidence interval, 1.1-4.4; P=.02). The macrolide-resistant strains were not identical. These findings suggest that macrolide resistance may be increasing in multiple strains in response to antibiotic pressure.