BACKGROUND: The 23S rRNA A2058G point mutation in Treponema pallidum is associated with macrolide antibiotic treatment failure. Its prevalence and potential association with a molecular subtype within the United States are unknown. METHODS: During 2007 to 2009, 11 clinics across the United States sent samples from genital ulcers to the Centers for Disease Control and Prevention. Molecular techniques were used to identify T. pallidum DNA sequences, the A2058G mutation, and subtype of T. pallidum. Accompanying epidemiologic information was abstracted from medical records. RESULTS: A total of 141 samples with T. pallidum were collected from individuals whose median age was 33 years (range, 13-68 years): 118 were male (69% reported as men having sex with men [MSM]). The A2058G mutation was carried in 75 samples (53%) with T. pallidum, with samples from MSM (versus women and other men) more likely carrying the A2058G mutation (65/82 samples versus 8/57 samples; prevalence ratio, 5.7; 95% confidence interval, 2.9-10.8). Of 98 strain-typed samples, 61 (62%) were the 14d9 subtype of T. pallidum, which was also associated with samples with T. pallidum from MSM (prevalence ratio, 3.5; 95% confidence interval, 1.9-6.5). However, among T. pallidum from MSM, the A2058G mutation was not associated with the 14d9 subtype. CONCLUSIONS: The A2058G mutation and 14d9 subtype of T. pallidum were present throughout the United States. Both were more commonly found in T. pallidum from MSM compared with women or other men but were not associated with each other. Treating syphilis with azithromycin should be done cautiously and only when treatment with penicillin or doxycycline is not feasible. Copyright © 2012 American Sexually Transmitted Diseases Association All rights reserved.