A molecular-based subtyping system for Treponema pallidum was used during an investigation of increasing syphilis in Maricopa County, Arizona. Genital ulcer or whole blood specimens from patients with syphilis were assayed by a polymerase chain reaction (PCR) amplification of a T. pallidum DNA polymerase I gene. Positive specimens were typed on the basis of PCR amplification of 2 variable genes. In all, 41 (93%) of 44 of ulcer specimens and 4 (27%) of 15 blood specimens yielded typeable T. pallidum DNA. Twenty-four (53%) of 45 specimens were subtype 14f; other subtypes identified included 4f, 4i, 5f, 12a, 12f, 14a, 14d, 14e, and 14i. Only 2 specimens were from epidemiologically linked patients. This investigation demonstrates that multiple subtypes of T. pallidum can be found in an area with high syphilis morbidity, although 1 subtype (14f) was predominant. Four typeable specimens were from blood, a newly identified specimen source for subtyping.